REVIEW ARTICLE
Masquerading microbial pathogens: capsular polysaccharides
mimic host-tissue molecules
Brady F. Cress1, Jacob A. Englaender2, Wenqin He1, Dennis Kasper3, Robert J. Linhardt1,2,4 &
Mattheos A.G. Koffas1,2
1
Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute,
Troy, NY, USA; 2Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA;
3
Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA; and 4Department of Chemistry, Chemical Biology,
Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA
Correspondence: Mattheos Koffas and
Robert Linhardt, Rensselaer Polytechnic
Institute, Troy, NY, USA.
e-mail: [email protected]
and [email protected]
Received 25 June 2013; revised 16 October
2013; accepted 19 December 2013. Final
version published online 27 January 2014.
DOI: 10.1111/1574-6976.12056
MICROBIOLOGY REVIEWS
Editor: Miguel Camara
Keywords
capsular polysaccharides; glycosaminoglycans;
polysialic acid; bacterial pathogens; immune
system evasion; combating antibiotic
resistance.
Abstract
The increasing prevalence of antibiotic-resistant bacteria portends an impending postantibiotic age, characterized by diminishing efficacy of common antibiotics and routine application of multifaceted, complementary therapeutic
approaches to treat bacterial infections, particularly multidrug-resistant organisms. The first line of defense for most bacterial pathogens consists of a physical and immunologic barrier known as the capsule, commonly composed of a
viscous layer of carbohydrates that are covalently bound to the cell wall in
Gram-positive bacteria or often to lipids of the outer membrane in many
Gram-negative bacteria. Bacterial capsular polysaccharides are a diverse class of
high molecular weight polysaccharides contributing to virulence of many
human pathogens in the gut, respiratory tree, urinary tract, and other host tissues, by hiding cell surface components that might otherwise elicit host
immune response. This review highlights capsular polysaccharides that are
structurally identical or similar to polysaccharides found in mammalian tissues,
including polysialic acid and glycosaminoglycan capsules hyaluronan, heparosan, and chondroitin. Such nonimmunogenic coatings render pathogens insensitive to certain immune responses, effectively increasing residence time in host
tissues and enabling pathologically relevant population densities to be reached.
Biosynthetic pathways and capsular involvement in immune system evasion are
described, providing a basis for potential therapies aimed at supplementing or
replacing antibiotic treatment.
Introduction
Bacterial capsular polysaccharides (CPSs) are major virulence factors that confer protective effects to their bearers against a wide range of environmental pressures,
most notably against the immune system during infection of their animal hosts. Although capsules are often
associated with descriptions of pathogenic bacteria due
to the large proportion of encapsulated invasive pathogens, nonpathogenic and commensal bacteria also benefit
from the ability to envelope themselves with a capsule
(Hafez et al., 2009; Dasgupta & Kasper, 2010). In Gramnegative bacteria, capsular polysaccharides are often
attached to the outer membrane at their reducing end
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
through covalently linked lipids that are inserted into
the lipid bilayer of the membrane. This provides a surface layer of water-saturated, high molecular weight
polysaccharides that limit desiccation in the face of
harsh environmental conditions, block infection by most
bacteriophages, and thwart phagocytosis and other host
immune responses by physically restricting access to cell
surface antigens. These polysaccharide cloaks are likely
rational targets for wide-spectrum therapeutic compounds aimed at replacing or supplementing antibiotic
treatment of microbial infections, as removal of the
capsule exposes bacteria to routine immune clearance
pathways mediated frequently by activation of the
complement system.
FEMS Microbiol Rev 38 (2014) 660–697
Masquerading microbial pathogens
Historical perspective
The molecular compositions of CPSs vary extensively
between organisms and even between strains within a single species, but, despite this diversity, some species from
distinct orders have been shown to biosynthesize identical
CPS structures (DeAngelis, 1999; DeAngelis & White,
2002). The existence of highly homologous biosynthetic
machinery for production of identical polysaccharides
between microorganisms suggests that capsular gene clusters have been acquired through horizontal gene transfer;
conversely, nonhomologous glycosyltransferases biosynthesize identical polysaccharides in disparate organisms
(Vann et al., 1981; Finne et al., 1983; Korhonen et al.,
1985; Jann & Jann, 1998), an occurrence that has likely
developed through functional convergent evolution
(DeAngelis, 2002a, b) facilitated by interkingdom coevolution of prokaryotic pathogens with their eukaryotic
hosts (Gagneux & Varki, 1999; Chen & Varki, 2010).
Capsule structure diversity was originally investigated as
part of a broad effort to classify bacterial strains based
upon their interaction with human serum; that is, bacteria were serotyped through differentiation of their cell
surface antigens (Lancefield, 1933). Serotyping is critical
for understanding pathogenicity from medical, diagnostic,
and immunologic perspectives and has remained the
dominant method for classifying strains of capsular bacteria. While studies in the early twentieth century demonstrated that polysaccharides present in the capsules of
both Gram-negative and Gram-positive bacteria were
antigenically distinct from cellular protein fractions (Heidelberger & Avery, 1923), research throughout the following decades further differentiated and classified these
polysaccharidic antigens and ultimately implicated the
eponymous CPSs in elevated serum resistance and inhibition of granulocytic phagocytosis (Peterson et al., 1978;
Horwitz & Silverstein, 1980).
Complementary serological and clinical studies during
the latter half of the twentieth century identified a subset
of streptococcal, staphylococcal, meningococcal, and Escherichia coli CPSs associated with increased virulence and
widespread incidence in severe bacterial infections, provoking investigation of the relationship between CPS
structure and immunogenicity (Robbins et al., 1974; Kaijser et al., 1977). Elucidation of the chemical structures of
various K-antigens (an alternative name for the CPS of
E. coli) and apparent demarcation based upon their physical properties prompted development of a K-antigen
classification system (Orskov et al., 1977) that was revised
over time to incorporate genetic and biomolecular evidence (Jann & Jann, 1997), ultimately being supplanted
by a more robust grouping scheme based on genetic, biochemical, and molecular criteria (Whitfield & Roberts,
FEMS Microbiol Rev 38 (2014) 660–697
661
1999). Serotyping systems for other species were
developed in a similar manner, but the relative ease of
Gram-negative CPS structural characterization and the
genetic tractability of E. coli enabled more rapid development of the E. coli antigen classification scheme. Owing
to their antigenicity in mammals, most CPS structures
elicit T lymphocyte-independent immune responses that
induce IgM antibody production but fail to stimulate
T-cell-dependent IgM-IgG switching, an important attribute to ensure long-lasting immunity (Weintraub, 2003;
Avci & Kasper, 2010). However, purified CPSs from some
of the most commonly isolated strains were determined
to be nonimmunogenic due to structural identity with
human glycans (Edwards et al., 1982; Johnson, 1991;
Herias et al., 1997). Capsule-deficient mutants of these
strains generally exhibit decreased virulence, persistence,
and serum sensitivity (Pluschke et al., 1983; Herias et al.,
1997). As discussed in greater detail later, antibody generation proved difficult against purified mammal-like bacterial CPSs composed of hyaluronan (HA), heparosan, or
certain congeners of unsulfated chondroitin or polysialic
acid (PSA).
It should be noted here that there are reports of antibodies generated against these CPSs under unique circumstances (Frosch et al., 1985; Jennings et al., 1985;
Kabat et al., 1986; Kr€
oncke et al., 1990; Finke et al.,
1991; Troy, 1992; Born et al., 1996). However, careful
consideration should be given to possible antigenic
determinants for antibodies generated in such experiments and whether access to the epitopes would result
in protective responses in vivo. If serum-accessible portions of these CPSs are identical to mammalian glycans,
it seems unlikely that antibodies could be elicited against
these ‘self’ epitopes. In some cases, antibodies were
raised in autoimmune animal hosts, where self-protection capacity was diminished due to immune dysregulation (Bitter-Suermann et al., 1986). Immune response by
healthy animal hosts requires other possible explanations
to clarify this paradox:
1 The CPS possesses an exposed antigenic determinant
not found in the corresponding mammalian glycan, such
as a deacetylated amino sugar or terminal unsaturated
bond generated by a lyase (a class of enzymes acting to
cleave acidic polysaccharides through an eliminase mechanism, in contrast to hydrolyzing glycosidases, Linhardt
et al., 1986) or some other nonself-chemical decoration.
2 CPS purification exposes a nonmammalian antigenic
determinant, like an anchoring moiety composed of a
phospholipid or a monosaccharide or oligosaccharide linker constituent not biosynthesized in mammals.
3 The antigenic determinant spans a self and nonselfdomain on the purified CPS, thereby cross-reacting with
the self-domain.
ª 2013 Federation of European Microbiological Societies.
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662
4 The antibody is not specific for the CPS but is crossreacting due to structural similarity to the true antigen.
In one example, a human IgM class antibody reactive
against PSA was shown to be nonspecific due to the
antibody’s reactivity with polynucleotides, a scenario
where cross-reactivity was enabled possibly due to similar surface charge distributions in these two negatively
charged biopolymers (Kabat et al., 1986). An IgG antibody against PSA was also isolated in autoimmune
NZB mice (Frosch et al., 1985). Nevertheless, evidence
supports the conclusion that nonimmunogenic bacterial
CPSs sharing structural identity with host glycans confer
an additional protective advantage over immunogenic,
nonhost CPSs.
Due to their poor immunogenicity, mammal-like CPSs
were originally considered nontypeable by traditional
means. However, ‘typing’ of some nonimmunogenic CPSs
was later achieved by screening strains against phages
only capable of attachment and subsequent infection
when a specific CPS is displayed on the bacterial surface,
such as E. coli K1- and K5-specific phages (Roberts et al.,
1986; Scholl et al., 2001). With increasing genetic characterization of CPS-producing strains, polymerase chain
reaction and sequencing-based methods such as multilocus sequence typing (MLST) can now be used for fast
and accurate molecular typing (Townsend et al., 2001;
O’Hanlon et al., 2004; Durso et al., 2005; Kong et al.,
2005; Zhu et al., 2012). Despite the utility of
antibody-based serotyping, confounding variables at the
immunologic level make molecular diagnosis an attractive
alternative.
B.F. Cress et al.
Bacterial glycans
Bacteria produce an array of carbohydrates that are not
limited to CPSs, and an understanding of these bacterial
glycans is critical for appreciating the role of the CPS in
the pathogenesis of infection. As depicted in Fig. 1,
Gram-negative bacteria possess an external layer of long
CPS chains that are covalently anchored by phospholipids
to the outer leaflet of the outer membrane, an asymmetric lipid bilayer with an external layer composed primarily
of lipid A (also known as endotoxin) and an internal
phospholipid layer. Anchored to the outer membrane by
lipid A, the lipopolysaccharide (LPS) serves as a hydrophilic barrier to natural hydrophobic antibiotics and is
composed of three regions: (1) lipid A, a highly conserved
region that possesses two phosphorylated, b-1,6 linked Nacetyl-D-glucosamine (GlcNAc) saccharide residues bearing variable numbers and lengths of fatty acyl chains; (2)
the core oligosaccharide, which can be subdivided into
the inner and outer cores. The inner core is covalently
bound to lipid A and possesses a species-dependent or
strain-dependent nonlinear oligosaccharide composed of
3-deoxy-D-mannooctulosonic acids (KDO), heptoses, and
some nonglycan components such as pyrophosphoethanolamine (PPEtn). The outer core is linked to the terminal heptose of the inner core and possesses a more
variable nonlinear structure of primarily hexose residues;
(3) the last region of the LPS is known in E. coli as
the O-antigen due to its distinct antigenicity from the
K-antigen. The O-antigen is a repetitive glycan that varies
in composition and length between species and strains,
Capsular
polysaccharide
Lipopolysaccharide
Outer membrane
Periplasm
Inner membrane
Fig. 1. Schematic cross-sectional representation of layers constituting the bacterial cell wall of a typical Gram-negative bacterium. The thick
external CPS layer conceals the bacterium to prevent desiccation, bacteriophage infection, complement-mediated killing, and
opsonophagocytosis. The black and white inset (top left) shows a quick-freeze, deep-etch scanning electron micrograph of the Gram-negative
organism Bacteroides thetaiotaomicron (Martens et al., 2009); this SEM image was originally published in The Journal of Biological Chemistry.
Martens EC, Roth R, Heuser JE & Gordon JI. Cover image. J Biol Chem. 2009; 284(27):cover. © the American Society for Biochemistry and
Molecular Biology.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Rev 38 (2014) 660–697
663
Masquerading microbial pathogens
but it is typically masked from the environment by the
K-antigen. Finally, an oligopeptide-cross-linked lattice of
alternating b-1,4 linked GlcNAc and N-acetylmuramic
acid (MurNAc) residues known as peptidoglycan is constrained within the periplasmic space between the outer
and inner (cytoplasmic) membranes. In many species,
peptidoglycan sugar moieties are further modified after
installation (Vollmer, 2008). Water-soluble b-glucan polymers known as membrane-derived oligosaccharides
(MDOs) are found near the inner membrane and are
decorated with negatively charged ethanolamine, phosphoglycerol, and succinyl groups. These highly charged
MDOs protect the inner cell membrane from low osmotic
conditions (Esko et al., 2009). Thus, glycans are critical
components of the cell wall that contribute to structural
integrity and interaction with the environment (Comstock & Kasper, 2006; Fig. 2). As Gram-positive bacteria
do not possess an outer cell membrane, the peptidoglycan
layer is thicker compared with Gram-negative bacteria.
Lipoteichoic acids anchor the inner layers of peptidoglycan to a glycolipid extending from cytoplasmic membrane, while wall-associated teichoic acids tether
additional outer layers together through covalent linkages
to MurNAc residues. CPSs biosynthesized by Gram-positive bacteria can be anchored to the inner membrane, to
oligopeptide cross-linkers within peptidoglycan, or to
GlcNAc residues in the peptidoglycan lattice (Hanson &
Neely, 2012).
In addition to LPS and CPS, many species of Gramnegative and Gram-positive bacteria also biosynthesize
and secrete an assortment of high molecular weight
glycopolymers known as exopolysaccharides, which have
long been considered determinants of biofilm physicochemical properties (Costerton et al., 1987). Surprisingly,
over the last decade, exopolysaccharides from certain
microorganisms have also been shown to inhibit biofilm
formation by other microbial species (Valle et al., 2006;
Kim et al., 2009; Nithya et al., 2010; Bendaoud et al.,
2011; Jiang et al., 2011). As an example of the diversity of
bacterial glycans, the glycocalyx of a strain of E. coli can
simultaneously possess six exopolysaccharides. In addition
to biosynthesis of covalently bound O-antigen and Kantigen, a linear heteropolysaccharide known as enterobacterial common antigen (ECA) is produced by all
members of the Enterobacteriaceae family and is also frequently bound to the outer membrane. ECA consists of a
conserved [?3)-a-Fuc4NAc-(1?4)-b-ManNAcA-(1?4)a-GlcNAc-(1?]n repeating trisaccharide unit that can be
bound by the reducing end of GlcNAc to the LPS core,
anchored to the outer membrane by a phosphate bridge
with diacylglycerophosphate, or secreted in a water-soluble, cyclized, and partially 6-O-acetylated (on GlcNAc)
form with polymerization degree typically between 3 and
6 trisaccharide repeats (Erbel et al., 2003; Fregolino et al.,
2012). Other exopolysaccharides, such as colanic acid
(known as M-antigen), are also secreted into the environment by many E. coli strains. Although only some colanic
acid (CA) remains loosely associated around the cell,
especially when constrained within a biofilm during suboptimal growth, an E. coli strain has been isolated in
which CA is ligated to the outer core of the LPS in place
of the O-antigen (Meredith et al., 2007). Another
(a)
(b)
O-Antigen
repeat unit
Outer core
Glycocalyx
Inner core
Fig. 2. Glycan-centric schematic of typical
Gram-negative cell wall components.
Membrane proteins and other cell wall
constituents are neglected for simplicity.
(a) Cell wall cross-section. (b)
Lipopolysaccharide. (c) Capsular
polysaccharide. Abbreviations are as follows:
Lyso-PG = lyso-phosphatidylglycerol,
GlcNAC = N-acetylglucosamine,
MurNAc = N-acetylmuramic acid,
GalNAc = N-acetylgalactosamine,
KDO = 3-deoxy-D-mannooctulosonic acid,
PPEtn = pyrophosphoethanolamine,
GlcA = glucuronic acid.
FEMS Microbiol Rev 38 (2014) 660–697
(c)
Outer membrane
Peptidoglycan
Membrane derived
oligosaccharides
K-Antigen
repeat unit
Poly-KDO
linker
Inner membrane
Lipid A
GlcNAc
Heptose
Glucose
Phospholipid
MurNAc
KDO
GlcA
Lyso-PG
GalNAc
PPEtn
Peptide crosslinker
Glucose substituted
with ethanolamine,
phosphoglycerol, or
succinyl group
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
664
exopolysaccharide of E. coli, commonly secreted by many
other bacteria as well, is the adhesin poly-b-1,6-GlcNAc,
a homopolymer that encourages adherence and biofilm
formation (Wang et al., 2004, 2005). Finally, E. coli and
other bacteria also biosynthesize and secrete an exopolysaccharide known as bacterial cellulose, or poly-b-1,4-glucan, as a component of the bacterial extracellular matrix
(Zogaj et al., 2001). It can be inferred that dynamic regulation of this suite of bacterial extracellular glycans allows
sampling of many glycocalyx states to adapt to a wide
range of environments (Meredith et al., 2007).
While Gram-positive CPS structures are diverse and
can be difficult to characterize, Gram-negative CPS structures are comparatively simple and have thus been more
amenable to categorization (Whitfield, 2006). In spite of
the depth and breadth of chemical and serological analysis of CPS structures, however, there remains a dearth of
evidence regarding linkage of Gram-negative CPS to the
cell. A very recent and fascinating report has resolved this
longstanding enigma for a class of model E. coli and Neisseria meningitidis strains, demonstrating that capsules
assembled through a common ATP-binding cassette
(ABC) transporter pathway are biosynthesized on a nearly
conserved lyso-phosphatidylglycerol anchoring moiety
through an oligo-KDO linker, presumably guiding the
translocation of such CPSs to the cell surface in a CPSand organism-independent manner (Willis et al., 2013).
CPSs in this category are known as Group 2 K-antigens
in E. coli, but all characterized N. meningitidis strains
possess a homologous transport system described below.
Despite the variety of bacterial glycans, CPSs comprised
of nonimmunogenic polysaccharides are of primary medical interest due to their conspicuous ability to evade the
immune system. Those sharing identity with human polysaccharides have been cataloged in a number of pathogenic species, but the most well-characterized CPS
structures are found in E. coli, N. meningitidis, Pasteurella
multocida, and Streptococcus pyogenes. It is interesting to
compare P. multocida, Avibacterium paragallinarum, and
E. coli as strains of all species have been found to produce
chondroitin and heparosan, while certain strains of
P. multocida also possess HA capsules. Although
P. multocida and A. paragallinarum are predominantly
animal pathogens rather than human pathogens, the ability of these microorganisms to produce identical nonimmunogenic capsules to E. coli through similar yet distinct
genetic and enzymatic processes warrants inclusion of the
species in this review. Moreover, the diseases caused by
these organisms in livestock pose economic threats and
cause concern regarding the contribution of antibioticladen livestock feed to the spread of antibiotic resistance.
Identical PSA capsules are also produced between different species, including meningitis-causing strains of E. coli
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
B.F. Cress et al.
and N. meningitidis, while HA is found in the capsules of
strep throat and necrotizing fasciitis-causing S. pyogenes
and P. multocida, the etiological agent of fowl cholera
and many other mammalian and bird diseases. Bacteria
containing these CPSs are compiled in Table 1 and are
included in the discussion where relevant.
Molecular mimicry and coevolution
In evaluating the ability of host-like CPSs to evade the
immune system, it is important to understand which host
tissues contain similar molecules and how pathogens
coevolved with humans to enable such mimicry. The first
capsule type addressed in this review is known as polysialic acid or PSA, consisting of N-acetylneuraminic acid
(Neu5Ac) monomers joined with various glycosidic linkages. PSA capsules produced by strains of K1 E. coli,
N. meningitidis serotype B, Moraxella nonliquefaciens, and
Mannheimia haemolytica (previously Pasteurella haemolytica) A2 are characterized by a-2,8 glycosidic linkages
(Adlam et al., 1987; Devi et al., 1991), while PSA produced by other microorganisms possess a-2,9 glycosidic
linkages (N. meningitidis serotype C strains) or alternating
a-2,8 and a-2,9 glycosidic linkages (K92 E. coli strains;
Glode et al., 1977; Lifely et al., 1986). In mammals, PSA
is an a-2,8-linked polysaccharide on neural cell adhesion
molecule (NCAM), which is found on the surface of neurons, glial cells, and natural killer cells, a type of lymphocyte functioning as an integral part of the innate or
nonspecific immune response (Rutishauser, 2008; Chang
et al., 2009). Increased surface polysialylation leads to
charge repulsion and is associated with decreased NCAM
adhesion in animals (Rutishauser et al., 1985) and resistance to phagocytosis by PSA capsular bacteria (King
et al., 2007). Structural identity of mammalian PSA with
E. coli K1 and N. meningitidis type B CPSs – particularly
embryonic NCAMs, which contain more than 50 a-2,8
Neu5Ac repeating units, compared with a much lower
degree of polysialylation in adult NCAMs (Jann & Jann,
1998) – contributes to the neuroinvasiveness of these
incredibly virulent neuropathogens (Robbins et al., 1974;
Sarff et al., 1975). The PSA capsule is thought to enable
traversal of the blood–brain barrier (Kim et al., 2003),
thus leading to high rates of morbidity and mortality in
neonatal meningitis and serious neurological conditions
in survivors of the disease (Kaper et al., 2004).
The other three host-like CPSs discussed in this review
are considered glycosaminoglycans (GAGs), or negatively
charged, linear polysaccharides identical to the backbones
of GAGs found in animals and composed of a repeating
core disaccharide unit, comprised of an uronic acid resi€k et al., 1984).
due linked to an amino sugar (H€
oo
Although the monomeric sugar precursors constituting
FEMS Microbiol Rev 38 (2014) 660–697
665
Masquerading microbial pathogens
Table 1. Pathogenic bacteria possessing nonimmunogenic CPSs that are identical to human and animal glycans
CPS
GAG
Organism*
Serotype/
capsule type
Polysialic acid
No
Escherichia coli
K1
Polysialic acid
Polysialic acid
No
No
Neisseria meningitidis
Moraxella nonliquefaciens
B
Polysialic acid
No
A2
Chondroitin†
Yes
Mannheimia
(formerly Pasteurella)
haemolytica
Escherichia coli
K4
Urinary tract infection,
diarrhea (human); diarrhea (bovine)
Chondroitin
Chondroitin
Yes
Yes
Pasteurella multocida
Avibacterium paragallinarum
Type F
Genotype I
Fowl cholera (avian)
Coryza (avian)
Heparosan
Yes
Escherichia coli
K5
Urinary tract infection (human)
Heparosan
Heparosan
Hyaluronan
Yes
Yes
Yes
Pasteurella multocida
Avibacterium paragallinarum
Streptococcus pyogenes
Type D
Genotype II
Pneumonia (porcine)
Coryza (avian)
Scarlet fever, pharyngitis (human)
Hyaluronan
Yes
Streptococcus equi
ssp. zooepidemicus
Hyaluronan
Yes
Hyaluronan
Yes
Streptococcus dysgalactiae
ssp. equisimilis
Streptococcus uberis
Hyaluronan
Yes
Streptococcus equi ssp. equi
Hyaluronan
Yes
Pasteurella multocida
Hyaluronan
Yes
Avibacterium paragallinarum
Disease(s) (organism)
Reference(s)
Meningitis, urinary tract infection,
diarrhea, septicemia (human)
Meningitis (human)
Endopthlamitis, sepsis, meningitis,
endocarditis (human)
Bovine respiratory disease (bovine)
Silver et al. (1988)
Septicemia, meningitis, endocarditis
and arthritis (bovine, porcine,
ovine, and canine)
Streptococcal toxic shock
syndrome (human)
Mastitis (bovine)
Type A
Upper respiratory tract
infection (equine)
Respiratory disease (bovine, feline)
Coryza (avian)
Finne et al. (1983)
Bøvre et al. (1983), Devi et al. (1991)
and Rafiq et al. (2011)
Adlam et al. (1987) and
Rice et al. (2007)
Orskov et al. (1985), Moxley &
Francis (1986) and
Rodriguez et al. (1988)
Rimler & Rhoades (1987)
Wu et al. (2010) and
Zhao et al. (2010)
Minshew et al. (1978) and
Zingler et al. (1990)
Ewers et al. (2006)
Wu et al. (2010)
Wessels et al. (1991) and Ralph &
Carapetis (2013)
Wibawan et al. (1999) and
Wei et al. (2012)
Calvinho et al. (1998) and
Hashikawa et al. (2004)
Almeida & Oliver (1993) and
Almeida et al. (2013)
Anzai et al. (1999)
Borrathybay et al. (2003b)
and Ewers et al. (2006)
Sawata & Kume (1983) and
Byarugaba et al. (2007)
*In recent years, there has been much confusion regarding delineation of Streptococcus species and subspecies due to imprecise, muddled, and
archaic classification systems (Jensen & Kilian, 2012). Similar scenarios occurred with Avibacterium and Mannheimia. Organism names are provided as originally reported unless a clear indication of misclassification was detected.
†
Fructosylated.
these core disaccharide units are conserved in GAGs, the
disaccharide units in the animal GAGs heparan sulfate
and chondroitin sulfate are not strictly repeating because
they are variably sulfated and acetylated within a single
chain. The dominant disaccharide unit in the polysaccharide and the distribution of specific disaccharide types,
glycosidic linkage configurations, molecular weight,
degree of sulfation and acetylation, and in some cases
degree of epimerization define the class of GAG and contribute to heterogeneity within each class. GAGs exhibit
their numerous biologic activities by interacting with proteins including growth factors, chemokines, and adhesion
molecules (Capila & Linhardt, 2002; Linhardt & Toida,
2004). The interactions between GAGs and pathogens can
also represent the first line of contact between pathogen
and host cell and are crucial to a pathogen’s invasive
FEMS Microbiol Rev 38 (2014) 660–697
potential (Kamhi et al., 2013). Symbolic representations
of bacterial CPS structures and related animal glycan
structures are illustrated in Fig. 3.
In particular, the CPSs produced by K4 E. coli and
P. multocida type F strains are related to CS, a class of
sulfated GAG characterized by a [?4) b-D-glucuronic
acid (GlcA) (1?3) N-acetyl-b-D-galactosamine (GalNAc)
(1?]n disaccharide repeat, where position and extent of
sulfation are tissue and organism dependent (Rodriguez
et al., 1988; Volpi, 2007; Volpi et al., 2008). While K4
CPS GlcA residues are substituted with bisecting b-fructofuranose units between C2 of fructose and C3 of GlcA,
the fructose is acid labile, and K4 CPS is thought to exist
as an unsubstituted backbone in certain low pH environments (Jann & Jann, 1990). Conversely, P. multocida type
F CPS is identical to the unsulfated chondroitin precursor
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
666
B.F. Cress et al.
Chondroitin
β4
β3
β4
β3
Chondroitin sulfate*
β4
RE
NRE
β4
OH
HO
HOOC
β4 β3
OR
O
OR
α4 β4
α4 β4
6
O
α4
NS3S
6S
2S
NS
O
O
3
R O
NHY
OH
O
O
O
O
OH
HOOC
OR6
OH
NHAc
O
O
O
O
O
R3O
O
2
R O
O
R2O
α4
O
OR2
O
O
β4
OR6
HOOC
RE
HOOC
O
HO
α4
α4 β4
O
NHAc
O
β4
NS
6S
O
HO
OR
OH
O
NHAc
α4
RE
6
OR4
O
OH
4
β4
NRE
NS
6S
4S/
6S
O
HOOC
O
O
O
O
NHY
NHAc
OH
Polysialic acid
Polysialic acid
α8 α8 α8 α8 α8 α8
α8 α8 α8 α8 α8 α8
RE
NRE
HOOC
O
O
OH
HOOC
OH
GalNAc
OH
AcHN
HOOC
O
HO
O
O
OH
AcHN
GlcNAc
O
HO
O
Bacterial
capsular
polysaccharide
Human
glycan
Hyaluronan
GlcA
IdoA
β4 β3
β4 β3
β4 β3
β4 β3
RE
RE NRE
HOOC
OH
O
HO
O
OH
OH
Neu5Ac
β4 β3
NRE
O
HO
OH
AcHN
OH
OH
β4 β3
RE
O
O
HO
Hyaluronan
NRE
HOOC
O
AcHN
0S/
2S/
6S
OR2
OH
HO
HOOC
HO
Heparosan
β3
O
HO
RE
OH
4S/
6S
O
β3
β3
O
β4
0S/
2S/
6S
HOOC
β4 β3
NRE
HO
β3
NRE
Fructosylated chondroitin
β4 β3
4S/
6S
0S/
2S/
6S
4S/
6S
NHAc
OH
β4
α4
RE
O
O
HO
β3
NRE
O
O
O
Heparan sulfate/Heparin†
β3
HOOC
O
O
NHAc
Fructose
O
HO
OH
O
OH
HO
O
O
O
NHAc
Acid-labile glycosidic linkage
Fig. 3. Symbolic representations and chemical structures of glycans described in this review. Nonimmunogenic bacterial CPSs and structurally
related animal glycans exhibited side-by-side to demonstrate similarity between backbones. In the case of chondroitin sulfate (CS) and heparan
sulfate/heparin, bacterial CPS structures are identical to precursors of the mature human glycans depicted here. Of note, a related GAG known
as dermatan sulfate also shares the unsulfated chondroitin backbone as a biosynthetic precursor, but, unlike CS, some glucuronic acid residues in
the chain are epimerized to iduronic acid. CS type B possesses iduronic acid residues, so it is sometimes classified as dermatan sulfate.
Conversely, HA and PSA structures are identical in microbial CPS and mature human glycans. *R2,4,6 = H or SO3 ; †R2,3,6 = H or SO3 , Y = SO3
or Ac (Ac = COCH3). Detailed disaccharide structures have been reported elsewhere (Sugahara & Mikami, 2007; Chang et al., 2012b).
of animal CS (DeAngelis et al., 2002). Considering the
limited patterns of O-sulfo group substitution within the
disaccharide-repeating unit of animal CS (Sugahara &
Mikami, 2007), it is apparent that the order of the sulfonation reactions is important in CS biosynthesis and that
O-sulfo groups in certain positions can preclude the
activity of downstream sulfotransferases (Schiraldi et al.,
2010). CS occurs in animals as an O-linked glycan chain,
covalently bound to serine residues of proteins, through a
specific tetrasaccharide linkage, resulting in glycoconjugates known as proteoglycans (Esko, 1999). This class of
GAG is found primarily in the extracellular matrix where
one or more CS chain is attached to an array of core proteins affording proteoglycans with various structural and
regulatory roles. Proteoglycans mediate a myriad of physiological interactions such as cellular recognition, communication, migration, adhesion, and proliferation (Thelin
et al., 2013).
The CPS produced by K5 E. coli strains such as Bi
8773-41 and the probiotic strain Nissle 1917 (Lodinova
Zaadnikov
a et al., 1992) and also by P. multocida type D
strains is composed of heparosan (Vann et al., 1981;
DeAngelis & White, 2002). Heparosan, comprised of [?
4) b-D-GlcA (1?4) a-D-GlcNAc (1?]n repeating
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
disaccharide units, is identical to the mammalian precursor for heparin and heparan sulfate (Ly et al., 2011;
Fig. 3). Heparan sulfate is typically found on the cell
membrane and in the extracellular matrix as a component
of proteoglycans (Gallagher, 1989). Heparin, a highly sulfated variant of heparan sulfate, is biosynthesized as an
intracellular proteoglycan, serglycin (Li et al., 2012). The
GAG heparin is a widely used anticoagulant pharmaceutical (Capila & Linhardt, 2002). Although the natural function of heparin is not well understood, its release from
the granules of mast cells is localized to damaged tissue
and contributes to wound healing and defense against
opportunistic infection (Zehnder & Galli, 1999). The
nontemplate-driven biosynthesis of heparan sulfate and
heparin affords a diverse range of disaccharide units from
a modest number of biosynthetic enzymes (Fig. 3).
The GAG known as hyaluronan or HA is structurally
identical in animals and in capsules of S. pyogenes groups
A and C, P. multocida type A, and some other species of
bacteria (DeAngelis, 1999). HA GAG consists of an
unmodified [?4) b-D-GlcA (1?3) b-D-GlcNAc (1?]n
disaccharide-repeating unit in both animals and bacteria.
In animals, this high molecular weight polysaccharide is
the predominant GAG in the extracellular matrix and is
FEMS Microbiol Rev 38 (2014) 660–697
Masquerading microbial pathogens
found in high quantities in skin, connective tissues, cartilage, synovial fluid, and the vitreous humor of the eye
€k et al., 1984; Dougherty & van de Rijn, 1992).
(H€
oo
The role of these host-like, or ‘self’, capsules enveloping
the surfaces of invasive pathogens seems quite clear:
molecular mimicry enables such pathogens to evade an
immune system that has learned to ignore self molecules
based on cell surface interactions. As glycans are ubiquitous on cell surfaces (Gallagher, 1989), they are likely the
first molecules contacted by pathogens that utilize cellular
adhesion during infection, so pathogens bearing these
capsules have an evolutionary advantage. The question
that remains is how humans and other animals have
evolved to combat these camouflaged pathogens and how
pathogens continually evolve to successfully colonize their
animal hosts. In a series of papers, Ajit Varki argues that
genetic evidence suggests pathogens – which evolve orders
of magnitude faster than their hosts due to horizontal
gene transfer, high mutation rates, fast growth, and short
life spans – develop self-CPSs through convergent evolution, where the glycosyltransferase genes responsible for
biosynthesizing the glycans in pathogen and host are not
homologous in most cases (Gagneux & Varki, 1999).
Considering that glycosyltransferases are highly conserved
within the host and yet biosynthesize highly diverse glycan structures and distributions in different cell types and
tissues, coupled with the combinatorial style of glycan
interactions and the ability to maintain functional specificity when a participating glycan is modified, Varki also
argues that sexual reproduction-enabled mutations in
host glycosyltransferases and subsequent change in glycan
profile allow these multicellular organisms to adapt to
pathogenic pressure. Furthermore, Varki speculates that
the coevolution of pathogens and their hosts has not only
tailored the diversity of glycan structures and expression
patterns, but that pathogenic pressures stemming from
host-like capsules contribute significantly to speciation of
multicellular organisms (Varki, 2006).
Immune response and clearance of
encapsulated pathogens
Both evasion of complement-mediated killing and failure
of being ingested by phagocytic cells enhance the virulence of the CPS. The polysaccharide capsules are effective physical barriers that protect the bacteria from
being killed. The fact that bacteria capsules are commonly hydrophilic and negatively charged diminishes
their removal through phagocytosis. The hydrophilic
nature causes high-level hydration and reduces the
surface tension at the phagocyte and bacterium interface
(Kuberan & Linhardt, 2000). Additionally, the negatively
charged polysaccharides on the bacterial surface repel
FEMS Microbiol Rev 38 (2014) 660–697
667
the negatively charged surface of phagocytes, thus
increasing the unfavorable interaction when phagocytosis
or complement-mediated lysis occurs (Moxon & Kroll,
1990; Kuberan & Linhardt, 2000). According to van Oss
and Gillman, the phagocytic cells such as polymorphonuclear leukocytes (PMNs), monocytes, and macrophages repel the encapsulated bacteria due to the net Lewis
AB repulsion between the hydrophilic outer layers (Klainer & Geis, 1975; van Oss et al., 1975), which reduce
the surface tension between the phagocytic cell and the
bacterium (Moxon & Kroll, 1990). For example, the cell
surface of Staphylococcus aureus became less hydrophilic
after removing the capsule and its phagocytic uptake
was enhanced (van Oss et al., 1975). A similar phenomenon was observed with the encapsulated strain of
Salmonella typhimurium, which resists phagocytosis, but
when unencapsulated, it is readily phagocytized (Cunningham et al., 1975). Noneffective contact can often lead
to the failure of phagocytic engulfment. More intuitive
is simple charge–charge repulsion between the negative
charge of the CPS and the glycocalyx of the phagocytic
cell. The more highly charged the CPS, the more
likely a bacterium is to avoid opsonophagocytosis
(Moxon & Kroll, 1990). Poor phagocytosis of a ‘smooth
surface’ may directly result from the physical surface
properties instead of biologic interaction of capsules
with phagocytic signaling and complement-mediated
molecules. Direct experimental testing of this hypothesis
remains challenging.
The interaction between the CPS of the bacterial surface and the host’s complement system is also a key
contributor to bacterial virulence. In the early stage of
the immune response, the control and defense mechanism of the host are contingent on the classic and alternative complement pathways. The classic pathway is
usually initiated by antigen–antibody binding. The C1
complement complex, which is a multimolecular protease consisting of three subunits C1q, C1r, and C1s, triggers the classical pathway of complement, first binding
to the aggregated antibody molecule, then sequentially
cleaving and activating the complement protein C4 and
proenzyeme C2 to form a C3 convertase, C2bC4b (Jann
& Jann, 1997). This process is regulated by C4-binding
protein C4bp (Roberts, 1996) and is usually retarded
during the encapsulated bacterial invasion. The C3 convertase then converts C3 to the activated C3b, which
will be deposited on the bacterial cell surface. This process is controlled by factors B and H of the alternative
pathway (Jann & Jann, 1997). The alternative pathway
can be activated in the absence of antibody binding to
the bacterial surface and therefore is very important in
immunity to encapsulated or unencapsulated bacteria.
In other words, the alternative pathway provides a way
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
668
for the immune system to kill bacteria in the blood in
the absence of specific antibodies. The alternative
pathway utilizes the serum protein C3b, which is then
activated by serum factor B, D, and properdin (Moxon
& Kroll, 1990), to form convertase C3bBb that amplifies
the complement cascade for more C3 conversions and
C3b deposition (Roberts, 1996). The activation of C3b
results in a ligand targeting specific receptors on PMNs
or macrophages. The binding of a C3b opsonized
microorganism to the complement receptor on PMNs
or macrophages initiates phagocytosis and ultimately
killing of the encapsulated bacteria. In addition, following C3b deposition, the sequential activations of C5 to
C9 form a membrane attack complex that directly leads
to the lysis and death of some Gram-negative bacteria
(Moxon & Kroll, 1990; Roberts, 1996).
This bacterial defense mechanism and the subsequent
response of complement-mediated bacterial killing by the
host can be blocked at numerous sites by CPSs avoiding
serum-mediated killing and enhancing virulence. Some
capsules protect the bacteria from being attacked by steric
mechanisms. Bacteria such as pneumococci promote C3b
deposition on the bacterial cell surface underneath the
capsule, shielding it from recognition by the phagocytic
cell (Winkelstein, 1981). Some bacterial capsules interrupt
the binding of C3b to the bacterial surface by affecting
regulatory proteins, such as factor B and H (Loos, 1985;
Cross, 1990). Capsules that exert such a defense mechanism usually contain N-acetylneuraminic acid (Neu5Ac)
because it contains a factor H binding site. The stimulation of H-C3b correspondingly decreases the amplification convertase C3bBb, leading to failure of the
complement cascade (Moxon & Kroll, 1990). Some capsules cannot bind to factor B, thus causing more H-C3b
formation (Winkelstein, 1981). Strains such as E. coli K1,
E. coli K92, N. meningitides types B and C, and Group B
Streptococcus polysaccharides have capsules that inhibit
alternative complement activation by these mechanisms
(Stevens et al., 1978; Wessels et al., 1989).
The mimicry of the CPS structure to substances within
the host serves as a virulence factor preventing bacteria
phagocytosis. A CPS can mimic a similar structure found
within the host representing ‘self’, and therefore, both
avoid recognition as foreign and circumvent triggering
the host immune response (Kuberan & Linhardt, 2000).
The CPS K1 has the same poly-a-2,8-Neu5Ac (PSA)
structure as carbohydrate portion of NCAM, required for
organogenesis and neural cell growth (Finne, 1982; Kuberan & Linhardt, 2000). Similarly, the CPS K5 strain of
E. coli shares the same structure as mammalian heparosan (Navia et al., 1983). An X-ray diffraction study
showed that the K4 capsule was poorly immunogenic
due to its similar helix structure to CS. The removal of
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
B.F. Cress et al.
fructosyl linkage under low pH environment transforms
K4 CPS into nonimmunogenic chondroitin (Jann & Jann,
1997).
Capsular polysaccharide transport,
genetics, biosynthesis, and role in
immune system evasion
The chemical properties and immunogenicity of CPSs are
dictated by variations in number, order, and diversity of
monosaccharide constituents, anomeric centers (a- or b-),
glycosidic linkage positions, absolute configuration (L or
D), ring forms (pyranose or furanose), degree of chemical
modification (O-acetylation, for example), and overall
conformation (Mazmanian & Kasper, 2006). There is a
wide range of capsule types among bacterial orders and
even within a single species. For instance, strains belonging to one of the most well-studied CPS-producing species, E. coli, are known to biosynthesize c. 80 CPS
structures. The number of known capsule types increases
dramatically when considering other genera, but capsules
in other organisms are less well characterized due to limited biochemical studies and relative genetic recalcitrance.
Nevertheless, studies in the model capsular species E. coli
suggest that the capsule assembly pathways are comparatively limited in scope, where a diverse assortment of CPSs
is assembled and translocated to the cell surface using
identical strategies. Biochemical and genetic evidence in
Gram-negative bacteria paints a picture of a veritable
orchestra of catalytic enzymes, structural proteins, and
transport proteins interacting in a transmembrane complex that spatially and temporally organizes biosynthesis
and transport. The modularity of the cooperating subcomplexes allows distinct CPS biosynthetic enzymes, complexed at the inner membrane, to utilize identical
transport systems for translocation of disparate CPS.
Whitfield and coworkers recently showed an ABC-transporter-dependent pathway common to some E. coli and
N. meningitidis strains results in the biosynthesis of
unique CPSs on a common anchor structure (Willis et al.,
2013). This apparently ensures successful CPS transport
and outer-membrane attachment. Similarly, another commonly conserved transport system, known as the Wzydependent pathway, shares the ability to assemble CPSs
with relaxed specificity for CPS structure. Although a wide
range of bacteria utilize the ATP-dependent and Wzydependent pathways for CPS assembly, the majority of
experimental evidence has been acquired in E. coli.
Homologous genes between species have been identified
by sequence similarity in many cases rather than by functional characterization. Hence, this section of the review
will focus on E. coli as a model system and draw comparisons between related bacteria where relevant.
FEMS Microbiol Rev 38 (2014) 660–697
Masquerading microbial pathogens
Transport pathways
In E. coli, CPS structures have been classified into four
groups. Group 1 and 4 CPS structures (as well as colanic
acid) are found in enteropathogenic (EPEC), enterotoxigenic (ETEC), and enterohemorrhagic E. coli (EHEC)
strains and are assembled through what is known as the
Wzy-dependent pathway. This pathway is distinct from
the so-called ABC-transporter-dependent pathway that is
responsible for assembly and transport of Group 2 and 3
CPSs and that is described in detail later. While uronic
acid sugars are common to Group 1 CPS repeat units,
Group 4 CPS repeats are characterized by the presence of
acetamido sugars. Despite this apparent structural distinction between Group 1 and 4 CPSs, both are polymerized
and transported to the cell surface in a similar manner.
In the Wzy-dependent system, serotype-specific repeating
units are assembled from cytosolic sugar precursors and
linked to undecaprenyl diphosphate by glycosyltransferases, unique to the specific type of CPS being synthesized,
which are embedded in the cytoplasmic membrane. Individual undecaprenyl diphosphate-linked repeating units
are then transferred across the inner membrane to the
periplasm by a flippase, Wzx, which also passes the repeat
unit to an integral membrane protein known as Wzy.
Wzy processively catalyzes addition of these individual
Group 1 and 4 CPS repeat units to the reducing end of
the growing polysaccharide chain, which elongates in the
periplasm without being released by Wzy until chain termination (Yi et al., 2006). Wza, Wzb, and Wzc are
responsible for control of chain length and export from
the periplasmic face of the inner membrane to the cell
surface. In Group 4 strains, longer polysaccharide chains
can be incorporated into the LPS structure and effectively
anchored by lipid A, although these K-antigens are classified as KLPS to distinguish their unique attachment mechanism (Whitfield, 2006). In Group 1 strains, shorter
polysaccharide chains can also form KLPS, but longer
chains are known to assemble capsules without covalent
attachment to LPS. Although the outer-membrane protein Wzi had been implicated in attachment of Group 1
CPSs (specifically the K30 antigen) to the outer membrane (Rahn et al., 2003), the exact mechanism was
unknown until recently. A paradigm shift in understanding CPS attachment resulted from a study that concluded
K30 CPS remained associated with the outer surface of
the cell due to interactions with an outer-membrane lectin, Wzi, that captures secreted CPS and serves as a
nucleation site for further CPS recruitment (Bushell et al.,
2013). Wzy-dependent capsules are also biosynthesized in
Klebsiella pneumoniae, and much of the molecular insight
for early steps in this pathway came from studies of Salmonella enterica O-antigen assembly. As CPSs in this class
FEMS Microbiol Rev 38 (2014) 660–697
669
do not share identity with animal glycans, they elicit an
immune response and are thus out of the scope of this
review. The reader is directed to two excellent reviews
compiling recent research in this area (Whitfield, 2006;
Reid & Cuthbertson, 2012).
Group 2 and 3 E. coli CPSs are produced in strains
commonly associated with extraintestinal infections
(ExPEC), while all known E. coli CPS structures sharing
identity with animal glycans belong to Group 2. It is also
interesting to note that Group 2 and 3 E. coli CPSs share
certain similar structure and assembly characteristics with
strains of N. meningitidis, P. multocida, Haemophilus
influenzae, and Campylobacter jejuni (Whitfield, 2006). In
contrast to Group 1 and 4 CPSs, the repeat units of
Group 2 and 3 CPSs exhibit extensive variation in structure. Similar to the Wzy-dependent transport system, the
ABC-transporter dependent system expressed by Group 2
and 3 E. coli strains has relaxed specificity for CPS structure, successfully transporting very distinct structures
across the cell wall. A striking difference compared with
Wzy-dependent assembly is that Group 2 and 3 CPSs,
assembled by ABC-transporter-dependent pathways, are
completely polymerized in the cytoplasm and then transported across the cell wall to the outside of the cell. CPSs
of this class are elongated by processive, CPS-specific glycosyltransferases that are colocalized to the cytoplasmic
surface of the inner membrane with other proteins
belonging to the coordinated biosynthetic-transport complex. Details regarding polymerization initiation are not
fully resolved, but CPSs from N. meningitidis group B,
E. coli K1, and E. coli K5 strains (all Group 2 type capsules) were recently shown (Willis et al., 2013) to be
linked to a well-conserved lyso-phosphatidylglycerol
(lyso-PG) terminus by a poly-b-KDO linker. It should be
noted that slight variation in fatty acyl chain length and
number of KDO repeats was measured within single cultures and between organisms. For instance, the single
fatty acyl chain of lyso-PG in most cultures varied
between saturated C16 (palmitoyl-PG) or monounsaturated C18 (oleoyl-PG), but one culture produced diacylPG with either two C16 chains (dipalmitoyl-PG), two
C18 chains (dioleoyl-PG), or one C16 adjacent to a C18
chain (palmitoyl-oleoyl-PG). Furthermore, the number of
KDO monomers exhibited interstrain and intrastrain variation between 5 and 9 KDO repeats. This discovery suggests that the common glycolipid carrier is the anchor by
which the ABC-transporter guides Group 2 CPSs from
the cytoplasm to the outer membrane. However, the
mechanism by which the glycolipid carrier is assembled
and attached to the nascent polysaccharide remains undetermined. Comparatively little is known about assembly
and transport of Group 3 CPS, but high sequence homology with Group 2 transport machinery suggests that the
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
670
two groups share a common transport mechanism. Studies on Group 3 strains, none of which are known to produce animal-like glycans, are reviewed elsewhere (Barrett
et al., 2002).
Genetics
Genes involved in CPS biosynthesis and transport are
typically organized within a so-called capsular gene cluster. Gene products participating in transport are generally
more well conserved, while capsule biosynthetic enzymes
are specific to capsule type, again suggesting an organization in which CPS transport proteins interface with CPS
biosynthetic enzymes in a modular, interchangeable fashion. In fact, episomal expression of CPS biosynthetic
enzymes from one Group 2 strain has been shown to lead
to functional capsule ‘transplantation’ in another acapsular Group 2 strain, where CPS is secreted by the common
transport complex as expected (Zhang et al., 2012). General characteristics of capsular gene clusters include distinct GC content compared with the rest of the
chromosomal DNA, lending additional evidence that
these genes were acquired through horizontal transfer.
Furthermore, CPS gene clusters are often encoded within
regions of the genome known as genomic (also pathogenicity-associated) islands (Sun et al., 2005; Wiles et al.,
2008), or segments of the genome prone to horizontal
gene transfer that often encode virulence factors (Ostblom
et al., 2011). Since the pioneering experiment in which
Silver and coworkers cloned and heterologously expressed
the E. coli K1 capsule – the first study to clone an entire
CPS gene cluster – Group 2 K-antigen assembly systems
have become the prototype for genetic and biochemical
characterization of ABC-transporter-dependent pathways
(Silver et al., 1981).
The gene cluster encoding biosynthesis and transport
of Group 2 capsules, including the capsule types of primary interest in this review, is depicted in Fig. 4a.
Regions 1 (kpsFEDUCS) and 3 (kpsMT) genes are conserved in Group 2 capsular E. coli and encode the
enzymes and transport proteins responsible for initiation
of chain elongation and translocation to the cell surface,
while Region 2 genes encode the glycosyltransferases and
other enzymes responsible for biosynthesis of the K-antigen-specific CPS. In comparison with Groups 1, 3, and 4,
expression of Group 2 CPS is subject to thermoregulation. Promoters upstream of Region 1 and Region 3 are
sufficient for transcription of all genes within the CPS
cluster at temperatures near the optimum of 37 °C, but
no Region 1 or 3 transcripts are detectable at temperatures below 20 °C (Cieslewicz & Vimr, 1996; Simpson
et al., 1996; Stevens et al., 1997; Whitfield & Roberts,
1999; Rowe et al., 2000; Xue et al., 2009). Specifically,
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
B.F. Cress et al.
kpsMT of Region 3 encodes the ABC transporter
responsible for translocation of the fully synthesized CPS
across the inner membrane (Reizer et al., 1992). The
transporter consists of multiple protein products, where
two units of KpsM function as the inner-membrane spanning domain and two units of KpsT serve as the nucleotide-binding domain (Pavelka et al., 1994; Pigeon &
Silver, 1994; Steenbergen & Vimr, 2008). Region 3 is
organized into a single transcriptional unit such that the
start codon of kpsT overlaps the stop codon of kpsM by
two base pairs, and it has been suggested that the two
proteins are translationally coupled to facilitate their
interaction at the inner membrane (Smith et al., 1990;
Pavelka et al., 1991). Upon binding of ATP, KpsT undergoes a conformational change that is conveyed to KpsM,
which then experiences a change in conformation to
enable transport of CPS using the energy gained from
ATP hydrolysis (Bliss et al., 1996).
Region 1 encodes genes implicated in biosynthesis of
the poly-KDO linker, as well as in translocation initiation
and transport through the periplasm. The genes kpsED
encode two proteins that receive the CPS from KpsMT
and transport it to the outer membrane (Wunder et al.,
1994; Rosenow et al., 1995a, b). Functional deletions of
kpsED lead to accumulation of CPS in the periplasm,
which supports the role of KpsED in CPS translocation
(Silver et al., 1988; Bronner et al., 1993; Pazzani et al.,
1993). KpsE has been described as an adaptor protein
that spans the periplasm to guide CPS from the ABC
transporter toward KpsD, a channel allowing CPS passage
through the outer membrane (Rosenow et al., 1995a, b).
The gene kpsF encodes D-arabinose 5-phosphate isomerase, a homotetramer (Meredith & Woodard, 2006) that
interconverts D-ribulose 5-phosphate and D-arabinose
5-phosphate with higher turnover toward D-arabinose
5-phosphate. The adjacent gene kpsU encodes CMP-KDO
synthetase, a dimer (Jelakovic et al., 1996) that converts
D-arabinose 5-phosphate provided by KpsF to nucleotideactivated CMP-KDO (Rosenow et al., 1995a, b). The
absolute roles of the cytosolic proteins encoded by kpsC
and kpsS are not entirely elucidated. Group 2 strains with
deletions of kpsC (DkpsC) or kpsS (DkpsS) accumulate
high molecular weight polysaccharide intracellularly,
implicating these two proteins in control of polymer
length as well as in translocation initiation (Larue et al.,
2011; Willis et al., 2013). The accumulating cytosolic
polysaccharide inside DkpsC and DkpsS K1 strains was
recently found to be nonlipidated (Willis et al., 2013)
despite conflicting past reports (Frosch & M€
uller, 1993;
Tzeng et al., 2005), suggesting that either KpsC or KpsS
might catalyze the transfer of CPS to the glycolipid
anchor. Given that most genes required for CPS biosynthesis and transport are typically located within the CPS
FEMS Microbiol Rev 38 (2014) 660–697
671
Masquerading microbial pathogens
(a)
Conserved
Region 1
Region 3
CPS-specific
Region 2
neuS neuE neuC neuA
kpsF kpsE kpsD kpsU kpsC kpsS
α-Neu5Ac
K1
kpsF kpsE kpsD kpsU kpsC kpsS
kfoG kfoF
K4
6
kfiC
K5
Region D
kfoB kfoA kpsT kpsM
kfoC
kfiB
(Fructosylated
Chondroitin)
1
kpsT kpsM
kfiA
β-GlcA
1
Region C
Region E
(Heparosan)
Region D’
Region B
Region A
rfbC rfbA rfbB galE ctrG
B
(c)
(PSA)
β-GlcA, β-GalNAc
kfiD
kpsT kpsM
3
kfoE kfoD IS2
α-GlcNAc
CPS-specific
neuB neuD
4
2
kpsF kpsE kpsD kpsU kpsC kpsS
(b)
Conserved
5
6
Conserved
csb
α-Neu5Ac
cssC cssB cssA ctrA
3
4
ctrC ctrD
tex
galE2 rfbB2 rfbA2 rfbC2 ctrE
ctrF
5
(PSA)
Region 3
Region 1
Region 2
CPS-specific
phyB
phyA
hyaE
hyaD
1
β-GlcNAc, β-GlcA
A
phyB
phyA
phyB
phyA
K4
hyaC
fcbE
fcbD
1
β-GalNAc, β-GlcA
F
D
ctrB
IS
2
fcbC
dcbE
dcbF
1
β-GlcA, α-GlcNAc
hyaB hexD hexC hexB hexA
(HA)
fcbB hexD hexC hexB hexA
2
(Chondroitin)
dcbC dcbB hexD hexC hexB hexA
2
(Heparosan)
Fig. 4. CPS gene loci in Gram-negative bacteria expressing ABC-transporter-dependent CPS assembly pathways. (a) Gene loci encoding enzymes
and transport proteins required for assembly of Group 2 Escherichia coli K-antigens K1 (polysialic acid), K4 (chondroitin), and K5 (heparosan).
Genes encoded by Regions 1 and 3 are well conserved within Group 2 E. coli strains and encode enzymes required for CPS translocation across
the cell wall, while Region 2 encodes CPS-specific glycosyltransferases and other biosynthetic enzymes. (b) Gene locus encoding proteins required
for assembly of Neisseria meningitidis serogroup B CPS. Region A encodes CPS-specific biosynthetic enzymes and varies between serogroups,
while Regions B–E are highly conserved in all N. meningitidis serogroups. Regions B and C encode CPS translocation proteins homologous to
genes in Regions 1 and 3 of Group 2 E. coli, Regions D and D’ encode LPS assembly genes, and Region E has no known function. (c) Gene loci
encoding Pasteurella multocida type A, D, and F CPS biosynthetic and transport proteins. Regions 1 and 3 encode translocation genes whose
functions are relatively well conserved in P. multocida, and Region 2 encodes CPS biosynthetic enzymes unique to the serotype specified.
Homologous interspecies transport genes are color coded and connected by bands of matching colors. Genes encoding glycosyltransferases are
illustrated as white arrows with black outline and glycosyltransferase activity denoted within (bifunctional glycosyltransferases are labeled as
found in nature, with N-terminal domain displayed at 5′ end of gene and C-terminal domain displayed at 3′ end of gene). UDP-glucose
dehydrogenase is frequently encoded in CPS-specific biosynthetic clusters and is indicated here with diagonal lines. Other genes with known and
putative homologs are designated with matching numbers. Note: genes and operons are not drawn to scale.
biosynthetic gene cluster, it is also possible that either
KpsC or KpsS is a b-KDO-polymerase catalyzing the biosynthesis of the poly-b-KDO-linker (Willis et al., 2013).
FEMS Microbiol Rev 38 (2014) 660–697
Further studies are required to determine the roles played
by KpsC and KpsS in biosynthesis and translocation initiation.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
672
Common to Region 2 are genes encoding the glycosyltransferases required for assembly of the K-antigen-specific CPS structure. Glycosyltransferases in Group 2
E. coli are processive and catalyze the addition of highenergy nucleotide-activated sugar monomers to the nonreducing end of the growing CPS. It should be noted that
identical CPSs in disparate bacteria, such as P. multocida,
are biosynthesized by nonprocessive enzymes (DeAngelis
et al., 2003). Studies have shown that Group 2 glycosyltransferases colocalize to the cytoplasmic side of the
inner membrane with other proteins from the CPS gene
cluster and that the proteins form a hierarchical transenvelope hetero-oligomeric complex to efficiently couple
CPS assembly and transport (Rigg et al., 1998). Also often
encoded in Region 2 with the glycosyltransferases are
enzymes that biosynthesize CPS precursors but that do
not actively participate in chain elongation (Cimini et al.,
2012). In certain instances, enzymes within the CPS biosynthetic gene cluster are predicted to duplicate the function of enzymes encoded elsewhere in the chromosome
(Mu~
noz et al., 1998). However, sequence divergence
between the copies suggests that there could be an advantage conferred by the extra copy. Spatial colocalization of
such duplicated enzymes with the CPS biosynthetic complex might ensure higher local concentrations of CPS precursors. Finally, CPS clusters possess genes with unknown
functions that do not appear necessary for CPS production (Krahulec et al., 2005), while other encoded proteins
lacking detectable catalytic activity have been shown to
associate with the biosynthetic complex and increase biosynthetic productivity, possibly by lending structural
integrity to the biosynthetic complex or by fostering protein–protein interactions (Hodson et al., 2000).
CPS biosynthesis in N. meningitidis is not as well characterized as in the more genetically tractable microorganism, E. coli. However, the genomes of representative
strains from all known serogroups have been sequenced,
and an ABC-transporter-dependent capsule assembly
pathway with homology to Group 2 and 3 E. coli strains
is conserved among all N. meningitidis capsular strains
(Harrison et al., 2013). Unique to each serogroup, of
course, are CPS biosynthetic enzymes for serotype-specific
polysaccharide production. Despite the homology of
many CPS transport genes between N. meningitidis and
E. coli, the two distinct CPS loci exhibit limited synteny.
Six regions known as A-D, D’, and E exist within the
CPS gene locus of N. meningitidis, occurring in the order
D-A-C-E-D’-B (Fig. 4b). CPS-specific biosynthetic genes
are encoded within Region A, and CPS transport proteins
are encoded within Regions C and B. Protein sequence
alignments have been used to identify CPS transport proteins in N. meningitidis, and a new gene nomenclature
has recently been proposed to ensure consistent
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
B.F. Cress et al.
descriptions between strains, where the names of all
N. meningitidis CPS transport genes begin with ‘ctr’ to
denote capsule transport (Harrison et al., 2013). The four
genes encoding the transmembrane complex ctrA, ctrB,
ctrC, and ctrD are adjacent to each other within Region C
of N. meningitidis, which contrasts the organization of
the E. coli homologs kpsD, kpsE, kpsM, and kpsT, respectively. It seems intuitive that the proteins required for
CPS translocation across the cell wall, including the ABC
transporter proteins (KpsM/CtrC and KpsT/CtrD), the
periplasm spanning adaptor protein (KpsE/CtrA), and the
outer-membrane protein (KpsD/CtrB), would be encoded
within a single operon as in N. meningitidis. However, it
is likely that ancient genomic rearrangements have led to
the separation of kpsED and kpsMT between two independent transcripts in E. coli. Neisseria meningitidis genes
ctrE (formerly lipA) and ctrF (formerly lipB) are encoded
in Region B and are homologs of E. coli genes kpsC and
kpsS, respectively. Despite the recent demonstration that
DkpsC and DkpsS E. coli K1 strains accumulate nonlipidated CPS (Willis et al., 2013), mutations in N. meningitidis genes ctrE and ctrF have been shown to lead to
intracellular accumulation of lipidated CPS (Tzeng et al.,
2005). This disparity could represent a slight variation
between species in an otherwise highly similar transport
system, where lipidation and translocation initiation
events are decoupled in N. meningitidis but intertwined
in E. coli. Further research differentiating between these
steps and assigning biochemical functions to CtrE and
CtrF will help resolve this discrepancy. Regions D and D’
encode duplicates of genes required for biosynthesis of
N. meningitidis LPS (Hammerschmidt et al., 1994), while
the function of Region E is unknown.
Pasteurella multocida also harbors a CPS gene locus
that shares significant homology with Group 2 E. coli and
N. meningitidis CPS gene loci (Fig. 4c). The topology of
P. multocida type A, D, and F gene clusters more closely
resembles that of E. coli, where a central CPS-specific
region is flanked by two regions, Region 1 and 3, coding
for translocation proteins. Specifically, Region 1 encodes
the transport genes hexA, hexB, hexC, and hexD that are
homologous to E. coli kpsT, kpsM, kpsE, and kpsD,
respectively, while Region 3 encodes phyA and phyB, homologs to kpsC and kpsS genes predicted to lipidate CPS
and initiate translocation (Chung et al., 1998). The genes
in both regions are highly conserved among members of
all five serogroups (A, B, D, E, and F), but P. multocida
type B and E CPS loci exhibit slight rearrangements in
gene order (Boyce et al., 2010). Region 3 gene lipA
(homologous to phyA) is instead located between Regions
1 and 2, whereas lipB (homologous to phyB) maintains
synteny with other serogroups due to its preserved location on the opposite side of Region 2 (Boyce et al.,
FEMS Microbiol Rev 38 (2014) 660–697
673
Masquerading microbial pathogens
2000a). For serogroups A and B, Region 1 genes hexA–D
are known as cexA-D. CPS-specific biosynthesis in all serogroups is guided by the variable enzymes encoded in
Region 2, including the synthases responsible for CPS
polymerization. Experimental validation of the putative
functions of most P. multocida Regions 1 and 3 proteins
is lacking, but a DhexA mutant strain was acapsular, supporting the role of HexA in CPS transport through the
cell wall (Chung et al., 2001).
which is then converted by the UTP:G1P uridylyltransferase galU to UDP-glucose through transfer of a uridylyl
group from UTP, releasing a pyrophosphate (PPi). The
second module consists of four enzymes catalyzing the
formation of UDP-N-acetylglucosamine (UDP-GlcNAc)
from fructose-6-phosphate (F6P). F6P-amidotransferase
encoded by glmS transfers an amine group from glutamine to F6P to form glucosamine-6-phosphate, which is
further isomerized to glucosamine-1-phosphate by phosphoglucosamine mutase (glmM). An acetyl group is then
transferred to glucosamine-1-phosphate to form N-acetylglucosamine-1-phosphate, a reaction catalyzed by glucosamine-1-phosphate N-acetyltransferase (glmU). The gene
glmU encodes a bifunctional enzyme that subsequently
transfers an uridylyl group from UTP to N-acetylglucosamine-1-phosphate, forming UDP-GlcNAc and releasing
PPi. Specific biosynthesis of CPS from these intermediates
and transport out of the cell in these model strains will
be described in greater detail within. It is important to
note here that these CPSs have been found in other species (Table 1), and it is expected that the rapid increase
in microbial genome sequencing projects will continue to
reveal disparate bacteria sharing related capsular gene
loci. As HA capsules are not known to exist in E. coli, the
genetic and biosynthetic description will be presented
later.
Biosynthesis
Several model E. coli strains possessing K1, K4, and K5
capsules have been sequenced, and their amino/nucleotide
sugar metabolism is well conserved with only slight
genetic and metabolic differences (Chen et al., 2006;
Shuting Lu et al., 2011; Cress et al., 2013a, b, c). The
conserved biosynthetic steps for these E. coli CPSs are
representative of many bacteria and are shown alongside
major competing metabolic pathways in Fig. 5, with CPS
intermediates boxed in black. Two activated UDP-sugar
intermediates are required for biosynthesis of the GAGlike CPSs, while only one of these two precursors is
required for biosynthesis of PSA, a non-GAG CPS. The
cytosolic reactions constituting these two intermediate
pathways act as sinks on upper glycolysis and can be considered as two distinct modules, represented by the two
branches in Fig. 5.
In the first module, glucose-6-phosphate (G6P) is converted to UDP-glucose by sequential action of two
enzymes, while UDP-glucose is further converted to
UDP-GlcA (the immediate GAG precursor) in GAG-producing K4 and K5 strains. Phosphoglucomutase (encoded
by pgm) isomerizes G6P to glucose-1-phosphate (G1P),
Polysialic acid
PSA is not a GAG, but like GAGs, it is an acidic, linear
polysaccharide found in vertebrate tissues. PSA is composed of repeating sialic acid (Neu5Ac) monomers, where
the glycosidic linkage configuration is organism dependent
and commonly found as either a-2,8 or a-2,9 linkages or a
PENTOSE PHOSPHATE PATHWAY
Glucokinase –
EC 2.7.1.2 (glk )
GLYCOLYSIS
Phosphoglucoisomerase –
EC 5.3.1.9 (pgi )
Glucose-6-P
Glucose
ATP
Fructose-6-P
Glutamine
ADP
Glutamate
Phosphoglucomutase –
EC 5.4.2.2 (pgm )
Glutamine-fructose-6-P
amidotransferase –
EC 2.6.1.16 (glmS)
Glucosamine-6-P
Phosphoglucosamine
mutase –
EC 5.4.2.10 (glmM)
WALL
POLYSACCHARIDE
Glucosamine-1-P
Glucose-1-P
Acetyl-CoA
CoASH
UTP
Fig. 5. Central biosynthetic pathway for CPS
precursor production in Escherichia coli. This
metabolic model is representative of early CPS
biosynthesis for many bacteria, including those
of interest in this review.
FEMS Microbiol Rev 38 (2014) 660–697
PPi
UTP-glucose-1-P
uridylyltransferase –
EC 2.7.7.9 (galU)
N-acetylglucosamine-1-P
UTP
Key:
Cofactors
Major competing
pathways
PEPTIDOGLYCAN
PPi
UDP-glucose
Glucosamine-1-P
N-acetyltransferase –
EC 2.3.1.157 (glmU)
UDP-N-acetylglucosamine1-P uridylyltransferase –
EC 2.7.7.23 (glmU)
UDP-N-acetylglucosamine
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
674
B.F. Cress et al.
combination of the two. In E. coli K1, N. meningitidis serogroup B, M. nonliquefaciens, and M. haemolytica A2,
PSA possesses the mammalian-like [?8) Neu5Ac (2?]n
structure seen in Fig. 3. Other strains possess immunogenic PSA capsules due to the nonanimal glycosidic linkages. For instance, sialic acid units in E. coli K92 CPS are
a-2,9 linked, and in N. meningitidis, serogroup C are alternating a-2,8 and a-2,9 linked.
Polysialic acid biosynthesis
Escherichia coli strains expressing K1 CPS share Regions 1
and 3 of the CPS gene cluster with other Group 2 capsular bacteria (Fig. 4a; Roberts, 1996). Region 2 consists of
6 genes specific for biosynthesis of K1 PSA. As depicted
in Fig. 6, the first committed step of PSA biosynthesis is
catalyzed by UDP-GlcNAc 2-epimerase (encoded by
neuC), which epimerizes UDP-GlcNAc to N-acetylmannosamine (ManNAc; Vann et al., 2004). NeuNAc synthase (neuB) then catalyzes the condensation of ManNAc
and phosphoenolpyruvate (PEP) to NeuNAc and inorganic phosphate (Pi), where three carbons from PEP
extend the monosaccharide from six to nine carbons (Annunziato et al., 1995). CMP-NeuNAc cytidyltransferase
(neuA) utilizes a single molecule of CTP to activate NeuNAc with the transfer of CMP, thereby releasing pyrophosphate (PPi; Silver et al., 1988). The processive sialic
acid polymerase, polysialyltransferase, encoded by neuS
sequentially adds NeuNAc to the nonreducing end of the
nascent PSA chain (Silver et al., 1988). Although it
UDP-N -acetylglucosamine
UDP
UDP-N -acetylglucosamine
2-epimerase –
EC 5.1.3.14 (neuC )
N -acetylmannosamine
PEP
Pi
N -acetylneuraminate
synthase –
EC 2.7.1.60 (neuB )
N -acetylneuraminic acid
CTP
PPi
CMP-N -acetylneuraminate
cytidylyltransferase –
EC 2.7.7.43 (neuA)
CMP-N -acetylneuraminic acid
CMP
Polysialyltransferase –
EC 2.4.99.8 (neuS )
POLYSIALIC ACID
Key:
Cofactors
Product
Fig. 6. Biosynthetic pathway for PSA production in Escherichia coli
K1.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
has been suggested that neuD encodes a Neu5Ac
O-acetyltransferase due to the presence of a hexapeptide
repeat motif characteristic of an acyltransferase superfamily (Vimr & Steenbergen, 2006), this possibility is unlikely
as K1 CPS is not O-acetylated in many strains encoding
neuD. Another study demonstrated heterodimerization
between NeuD and NeuB, suggesting that NeuD plays a
stabilization role during chain elongation (Daines & Silver, 2000). The exact function of NeuE is unknown, and
further efforts will be required to understand its role in
polymerization initiation (Reid & Cuthbertson, 2012).
Many K1 CPS strains have been found to possess PSA
that has been O-acetylated at the C7 or C9 hydroxyl
group (Orskov et al., 1979). These are chemical modifications that increase resistance to desiccation and reduce
biofilm formation, while coincidentally increasing immunogenicity (Mordhorst et al., 2009). However, the O-acetylation is a dynamic phenotype that varies within a
population and appears to be controlled by a stochastic
‘on–off’ switching mechanism at the genetic level (King
et al., 2007). It was determined that this phase variation
(also known as form variation) only occurs in K1 strains
that have been lysogenized by a K1-specific lambdoid
bacteriophage and thus possess a chromosomal accretion
element known as CUS-3, a remnant of the infection.
The neuO gene, encoding the K1 O-acetyltransferase
responsible for the chemical modification of PSA, is
encoded within CUS-3. The 5′ end of neuO is subject to
slip strand DNA mispairing in which a 7 nucleotide
repeat sequence is gained or lost at the 5′ end, leading to
a frameshift and corrupted translation. By this mechanism, individuals in a CUS-3-harboring K1 population
randomly partition between acetylation-on and acetylation-off variants, a phenomenon that presumably confers
a population-level evolutionary advantage where the likelihood of persistence increases in adverse environmental
pressures (Deszo et al., 2005). Incredibly, the O-acetyltransferase catalytic efficiency has also been shown to
increase linearly with the number of tandem, in-frame
repeats that manifest as tandem heptapeptide repeats and
form a disordered N-terminal domain (Schulz et al.,
2011). The function of the disordered region remains
unknown. PSA purified from K1 strains lacking the CUS3 region was invariably lacking O-acetylation (Deszo
et al., 2005).
Neisseria meningitidis serogroup B CPS is identical to
K1 CPS, although the genes encoding the biosynthetic
enzymes are organized differently and share only 30–40%
amino acid sequence identity. Neisseria meningitidis serogroup B Region A encodes cssABC (formerly synABC or
siaABC), csb (formerly synD or siaD), and ctrG (formerly
NMB0065; Fig. 4b). The cssA gene encodes an UDP-GlcNAc 2-epimerase that shares 32% identity with NeuC
FEMS Microbiol Rev 38 (2014) 660–697
Masquerading microbial pathogens
from K1 (Murkin et al., 2004), while cssB codes for a
CMP-Neu5Ac cytidylyltransferase with 34% identity to
K1 NeuA (Edwards & Frosch, 1992; Ganguli et al., 1994).
The protein encoded by cssC shares 37% identity with its
K1 homolog NeuB, a Neu5Ac synthase (Vimr et al., 1989;
Ganguli et al., 1994). The csb gene codes for a polysialyltransferase with 33% identity to K1 NeuS (Frosch et al.,
1991). Finally, the gene products of ctrG and K1 neuE
share 27% identity. The role of CtrG in polymerization
initiation is not entirely elucidated, but is appears to play
a similar role as NeuE in coupling CPS biosynthesis with
traversal through the cell wall (Hobb et al., 2010). Thus,
the pathways biosynthesizing polysialic acid in these two
Gram-negative organisms are metabolically and functionally identical. Furthermore, protein homology demonstrates high functional conservation of the ATPdependent transport pathways. Biosynthesis of a-2,8-PSA
capsules in other species, including M. haemolytica A2
(Adlam et al., 1987) and M. nonliquefaciens (Devi et al.,
1991), has not been studied in depth.
Polysialic acid in evasion of immune
system
Compared with other CPSs, K1 E. coli strains are particularly nonimmunogenic due to the fact that the structure
of CPS mimics the substance in the host (Brooks et al.,
1980). The chemical structure of K1 polysaccharide is
identical to the PSA on the embryonic form of NCAM,
which is related to the organization of the neural tissue
(Finne et al., 1983). Therefore, they are relatively more
virulent because the immune response toward K1 is usually nonexistent due to the mistaken recognition of the
encapsulated bacteria as ‘self’, letting them pass protective
barriers. Interestingly, this only applies to a certain host
age range. The K1 organism’s carriage rates are 22–42%
among infant and children without sex differentiation,
while the highest among women aged 16–31 years old
(Sarff et al., 1975). Some studies also indicate that K1
strains are poor activators for initiating the alternative
complement pathway of immune response (Bortolussi
et al., 1979). The anticomplementary effect is due to
PSA’s increasing the binding of inhibitor B1H to C3b,
preventing formation of C3 convertase, and blocking activation of the complement cascade (Harber et al., 1986;
Leying et al., 1990). The failure to accumulate C3b on
the cell surface effectively prevents phagocytosis and, thus,
enhances the virulence of E. coli K1 strain.
Escherichia coli K1 strains frequently cause infections of
the urinary tract (Johnson, 1991), which, according to
Wiles, is one of the most common sites associated with
human disease (Wiles et al., 2008). Moreover, K1 is also
mainly responsible for causing acute pyelonephritis
FEMS Microbiol Rev 38 (2014) 660–697
675
(Kaijser, 1973) as they can be easily found among the
bacterial strains isolated from clinical specimens during
acute pyelonephritis (Hanson et al., 1977). In addition,
the K1 antigen is also found on strains of the extraintestinal pathogenic E. coli (ExPEC; Wiles et al., 2008). K1
E. coli are generally thought to be the second most common cause of human neonatal meningitis (next to group
B streptococci), and c. 80% of American and European
strains implicated in the disease have PSA capsules (Orskov & Orskov, 1992). Some studies have also suggested
that bacterial survival within brain microvascular endothelial cells was enhanced by K1 CPS (Pluschke et al.,
1983; Kim et al., 2003; Scholl et al., 2005).
PSA is also the CPS of two serogroups of N. meningitidis, a common causative agent of meningitis in children
and adults. Early work on the CPS of N. meningitidis serogroup B found that mutants deficient in capsule production lost all pathogenicity in mice (Masson et al.,
1982). Several studies have demonstrated that the CPS
aids in the resistance of the bacterial cells to the innate
immune system (Jarvis & Vedros, 1987; Spinosa et al.,
2007). Similar to other CPSs, the PSA capsule of N. meningitidis has been found to hinder adhesion and invasion
(Spinosa et al., 2007), and it is unable to activate the
complement pathway (Jarvis & Vedros, 1987). This is
most likely a result of the capsule masking immunogenic
adhesins and invasins on the surface of the bacterial cell.
However, it has also been shown that the CPS is vital for
the survival of the bacterium in the bloodstream (Jarvis &
Vedros, 1987) and important for survival inside human
cells (Spinosa et al., 2007). In the bloodstream, the CPS
allows N. meningitidis to evade uptake and degradation
by macrophages (Jarvis & Vedros, 1987), while intracellularly, the encapsulated bacterial cells are resistant to antimicrobial peptides, which act by binding bacterial
membranes and increasing their permeability (Spinosa
et al., 2007).
Several studies have shown the ability to successfully
produce antibodies protective against N. meningitidis serogroup B by immunization with a vaccine containing Npropionyl and de-N-acetylated sialic acid derivatives (Pon
et al., 1997; Granoff et al., 1998; Moe et al., 2009).
Importantly, the antibodies were shown to be unreactive
to human PSA. More specifically, vaccines containing deN-acetylated sialic acid derivatives were shown to possess
the ability to protect against N. meningitidis in multiple
ways, including complement-dependent bactericidal activity and passive protection in infant mice (Moe et al.,
2009). Because the vaccines show protection against
N. meningitidis serogroup B, but not purified human
PSA, it is possible that some amount of de-N-acetylated
sialic acid is present in the CPS of N. meningitidis serogroup B. It has been proposed that de-N-acetylated sialic
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
676
B.F. Cress et al.
acid elicits a T-cell-dependent immune response due to its
zwitterionic nature, characterized by the presence of both
positively charged free amino groups at de-N-acetylated
positions and negatively charged carboxyl groups along
the polymer backbone (Moe et al., 2009). In contrast to
the more common negatively charged or neutral CPSs,
zwitterionic CPSs are known to be T-dependent antigens,
which are bound, processed, and presented by major
histocompatibility complex class II (MHCII) to stimulate
helper T-cells through what is known as the MHCII
endocytic pathway (Cobb et al., 2004; Surana & Kasper,
2012). These remarkable findings suggest that enzymatic
de-N-acetylation of other acidic CPSs such as heparosan,
chondroitin, and hyaluronan could represent a strategy
for eliciting natural immune response to pathogenic
infection.
Chondroitin
CPS produced by strains of K4 E. coli and P. multocida
type F is structurally related to the GAG CS, which is
involved in a range of molecular interactions in humans
as previously described. As loss of CS from cartilage in
humans leads to osteoarthritis, nutritional supplementation is a common treatment strategy, making CS a pharmaceutically and nutraceutically valuable product (Wildi
et al., 2011). CS is currently harvested from animal
sources (cow, pig, shark, fish, and bird cartilage; Huskisson, 2008), but there is a growing interest in moving to
sustainable microbial production platforms to minimize
contamination and to control product consistency. One
such production strategy involves harvesting a CS precursor from cultures of E. coli strains biosynthesizing a K4
capsule (Zanfardino et al., 2010; Restaino et al., 2011,
2012; Schiraldi et al., 2011, 2012; Trilli et al., 2012). This
biotechnological relevance has provoked increased interest
in improving biosynthesis of K4 CPS by manipulating
E. coli metabolism (Restaino et al., 2012; Cimini et al.,
2013; Cress et al., 2013b; Wu et al., 2013). K4 CPS is
UDP-glucose
2NAD+
Chondroitin polymerase/
glycosyltransferase –
EC 2.4.1.- (kfoC )
Escherichia coli strains expressing the K4 capsule share
Regions 1 and 3 of the Group 2 CPS biosynthetic cluster,
but the biosynthetic enzymes unique to K4 CPS are
encoded by Region 2 and presumably form a biosynthetic
complex at the cytosolic side of the inner cell membrane
(Fig. 4a). Biosynthesis of K4 CPS precursors can be segmented into two distinct modules drawing from upper glycolysis through the intracellular pool of UDP-glucose and
UDP-GlcNAc (Fig. 7). In the first module, UDP-glucose is
converted to UDP-GlcA by UDP-glucose dehydrogenase
(UGDH) encoded by kfoF and associated with the K4 CPS
biosynthetic enzyme complex (Ninomiya et al., 2002).
Although it is not uncommon for multiple copies of UDPglucose dehydrogenase to exist in E. coli genomes, the existence of two other copies in the genome of the model K4
strain U1-41 (Cress et al., 2013b) suggests that kfoF has
evolved to perform a distinct physiological role. As UDPglucose is a key metabolite in many pathways, it is plausible
that the association of the kfoF-encoded copy of UDP-glucose dehydrogenase with the capsular biosynthetic enzyme
complex serves to spatially constrain the chemical reaction –
conversion of UDP-glucose to UDP-GlcA – near the K4
CPS glycosyltransferase on the inner cell membrane,
increasing the local concentration of UDP-GlcA and effectively channeling valuable UDP-glucose toward production
of K4 CPS without significant loss to other cellular reactions. In the second module, UDP-GlcNAc 4-epimerase
encoded by kfoA catalyzes the formation of UDP-GalNAc
(Ninomiya et al., 2002). The two activated sugar precursors
UDP-N -acetylglucosamine 4epimerase –
EC 5.1.3.7 (kfoA)
2NADH + 2H+
UDP
Chondroitin biosynthesis
UDP-N -acetylglucosamine
UDP-glucose dehydrogenase –
EC 1.1.1.22 (kfoF )
UDP-glucuronic acid
similar in structure to unsulfated CS, with the exception
of an acid labile, bisecting b-fructofuranose attached to
C3 of the GlcA residue (Fig. 3). Alternatively, the
P. multocida type F CPS is a linear polysaccharide identical to unsulfated chondroitin (DeAngelis et al., 2002).
Commercial interest in this GAG has increased knowledge
regarding its biosynthesis in microorganisms.
UDP-N -acetylgalactosamine
UDP
Chondroitin polymerase/
glycosyltransferase –
EC 2.4.1.- (kfoC )
UNFRUCTOSYLATED CHONDROITIN BACKBONE
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
Key:
Cofactors
Product
Fig. 7. Biosynthetic pathway for production
of K4 CPS, chondroitin, in Escherichia coli K4.
FEMS Microbiol Rev 38 (2014) 660–697
677
Masquerading microbial pathogens
from each module are sequentially added to the nonreducing end of the growing polysaccharide chain by chondroitin
polymerase (encoded by kfoC), a bifunctional glycosyltransferase catalyzing the transfer of both GlcA and GalNAc
and release of two molecules of UDP per disaccharide
extension (Ninomiya et al., 2002). The crystal structure of
KfoC has been obtained in the presence of UDP-activated
precursors, indicating the existence of two active sites for
addition of UDP-GalNAc by the N-terminal domain and
UDP-GlcA by the C-terminal domain (Osawa et al., 2009).
Several studies have suggested that K4 chondroitin is
fructosylated subsequent to polymerization of the backbone (Lidholt & Fjelstad, 1997), but the enzyme responsible for fructosylation has remained unreported until
recently. Initial searches for the fructosyltransferase focused
on proteins in Region 2 of the K4 biosynthetic gene cluster
that were predicted to possess glycosyltransferase motifs.
One such enzyme encoded by kfoG possessed a putative
glycosyltransferase domain; however, disruption/deletion
of the gene did not prevent fructosylation (Krahulec et al.,
2005). A series of recent patents purport that deletion of
kfoE results in production of unfructosylated chondroitin,
suggesting that kfoE encodes a fructosyltransferase (Trilli
et al., 2012). The enzyme has not been characterized in its
purified form, however, and biochemical characterization
will be required to assign fructosyltransferase activity to
kfoE. Several genes in the K4 CPS cluster, including kfoB,
kfoD, and kfoG, have unknown function. However, kfoB
shares 38% identity with a gene in E. coli K5 (kfiB) that is
believed to be structurally important in K5 CPS biosynthesis. This might suggest a stabilizing role for KfoB that is
critical for assembling or colocalizing biosynthetic enzymes
near the transenvelope CPS assembly complex, but further
work will be required to determine the precise functions of
these proteins.
The unfructosylated, unsulfated chondroitin backbone
constituting the P. multocida type F CPS is biosynthesized
by genes in Region 2 of the P. multocida CPS locus. The
region consists of four genes, fcbB, fcbC, fcbD, and fcbE,
only one of which has been characterized. The chondroitin synthase known as PmCS is encoded by fcbD and is
homologous to E.coli K4 kfoC (DeAngelis et al., 2002).
PmCS is also a bifunctional polymerase possessing two
distinct glycosyltransferase domains that sequentially add
GlcA and GalNAc residues to the growing polysaccharide
chain (DeAngelis & Padgett-McCue, 2000). Similar to
KfoC, the N-terminal domain of PmCS possesses GalNAc-transferase activity and the C-terminus possesses
GlcA-transferase activity (Osawa et al., 2009; Otto et al.,
2012). While deletion of kfoE in E. coli K4 has been
reported to abrogate chondroitin fructosylation, it would
be interesting to determine whether heterologous
expression of kfoE in P. multocida type F complements
FEMS Microbiol Rev 38 (2014) 660–697
expression of fructosylated CPS. Pasteurella multocida
genes fcbB, fcbC, and fcbE are homlogous to E. coli K4
genes kfoG, kfoF, and kfoB, respectively, but the biologic
functions have not been studied (Townsend et al., 2001).
Chondroitin in evasion of immune
system
The K4 antigen is not implicated in human disease as frequently as K1 and K5 antigens, but K4 strains have been
associated with human and animal infection. K4 E. coli
strain U1-41 is a uropathogen (Rodriguez et al., 1988),
and the K4 capsule has been found on strains causing
diarrhea in humans (Orskov et al., 1985). EHEC K4
strains have also been isolated in calves (Moxley & Francis, 1986; Stordeur et al., 2000). The unfructosylated
chondroitin backbone is identical to the mammalian CS
precursor, but unlike the K1 and K5 antigens, the K4
antigen host-related bacterial polymer is substituted with
an additional sugar residue not present in the mature,
sulfated GAG. The b-linked fructose on K4 CPS is an
antigenic determinant that imparts immunogenicity to
the E. coli K4 capsule and is responsible for a conformational change resulting in significantly higher viscosity
compared with defructosylated K4 CPS (Rodriguez et al.,
1988). Therefore, K4 CPS may be easily recognized by
anti-K4 antibodies and induce a complement-dependent
immune response. However, the fructosyl group is labile
and can be easily removed in mild acidic conditions. The
conversion of K4 antigen to a nonfructosylated chondroitin results in the nonimmunogenicity of K4 CPS (Rodriguez et al., 1988; Jann & Jann, 1997). Thus, it has been
suggested that such lability enables dynamic physiological
conditions in the host, such as low pH in certain host tissues, cells, or compartments (Jann & Jann, 1997), to
modulate the presence of this immunodominant residue
on the K4 capsule. A K4 strain also might benefit outside
the host from a more viscous capsule and inside the host
from a less viscous, nonimmunogenic capsule.
The chondroitin CPS of P. multocida type F is nonimmunogenic as it lacks the fructose residue present in
E. coli K4 CPS and is thus identical to the animal precursor to CS. It has been shown that treatment of P. multocida type F with chondroitinase, a lyase that is known to
cleave unsulfated chondroitin, results in increased phagocytosis by swine neutrophils (Rimler et al., 1995), presumably through elimination of the capsule.
Heparosan
Pharmaceutical grade heparin has traditionally been
derived from porcine or bovine mucosal tissues, but a
contamination crisis leading to several hundred deaths
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
678
B.F. Cress et al.
worldwide and causing severe allergic reactions in many
patients prompted exploration of microbial heparosan
production followed by enzymatic conversion to bioengineered heparin with purity and biologic activity meeting
United States Pharmacopeia (USP) standards (Wang
et al., 2011). Escherichia coli K5 strains have been shown
to serve as a viable production platform due to the similarity between K5 CPS and the mammalian precursor for
heparin (Wang et al., 2010, 2011; Ly et al., 2011). Recent
work has also demonstrated the ability of an engineered
heparosan-producing E. coli BL21 strain to naturally
secrete the polysaccharide (Zhang et al., 2012), presumably utilizing transport proteins encoded by Regions 1
and 3 genes from its endogenous Group 2 capsular export
system (Andreishcheva & Vann, 2006). This work demonstrated the feasibility of producing heparosan in wellcharacterized production strains, albeit at much lower
concentrations than wild-type K5 strains. Efforts in this
area have contributed to the understanding of heparosan
biosynthesis.
Heparosan biosynthesis
Similar to K4 CPS, the biosynthesis of K5 CPS can be
partitioned into two modules each producing the requisite activated sugar precursor for heparosan polymerization. The monomeric constituents of K5 CPS are GlcA
and GlcNAc, which draw carbon from G6P and F6P in
the form of two activated sugar precursors, UDP-GlcA
and UDP-GlcNAc, respectively. UDP-GlcA biosynthesis in
K5 is identical to that in K4 with the exception of the
UDP-glucose dehydrogenase associated with the capsular
biosynthetic complex. Conversion of UDP-glucose to
UDP-GlcA in the first module is catalyzed by an UDPglucose dehydrogenase encoded by kfiD from Region 2 in
the K5 capsule gene cluster (Fig. 4a; Sieberth et al.,
1995). The kfiD gene encodes the third copy of UDP-glucose dehydrogenase in the genome of the model strain Bi
8337-41 (Cress et al., 2013c) and the probiotic strain
UDP-N -acetylglucosamine
UDP-glucose
2NAD+
Nissle 1917 (Cress et al., 2013a), and biochemical studies
provide evidence for its association with the K5 CPS biosynthetic complex also comprised of proteins encoded by
kfiA, kfiB, and kfiC (Rigg et al., 1998). While kfiB is
thought to perform a membrane-localization and complex-stabilizing or scaffolding role rather than a catalytic
one (Hodson et al., 2000; Zhang et al., 2012), kfiA and
kfiC are glycosyltransferases that alternatively elongate the
heparosan chain at the nonreducing end (Petit et al.,
1995). The final reaction of the first module is catalyzed
by kfiC-encoded UDP-GlcA glucuronosyltransferase,
which attaches a GlcA residue to the nascent K5 polysaccharide and releases UDP. The second module in K5 CPS
biosynthesis encompasses the reactions converting F6P to
UDP-GlcNAc as shown in Fig. 8, and the final reaction
involves transfer of GlcNAc from UDP-GlcNAc to the
growing polysaccharide by kfiA-encoded UDP-GlcNAc Nacetylglucosaminyltransferase, a step that frees an additional UDP.
Relatively, little is known about most of the proteins
encoded in Region 2 of P. multocida type D (dcbB, dcbC,
dcbE). An exception is the well-studied heparosan synthase PmHS1 encoded by dcbF, also known as hssA (Kane
et al., 2006). This bifunctional synthase possesses two
fused glycosyltransferase domains, where a segment of the
N-terminal b-1,4-glucuronosyltransferase domain is
homologous to E. coli KfiC, and a section of the C-terminal a-1,3-N-acetylglucosaminyltransferase domain is
homologous to KfiA (Kane et al., 2006; Otto et al., 2012).
Drawing analogy with other CPS biosynthetic gene clusters, the putative UDP-glucose dehydrogenase encoded by
dcbC likely supplies UDP-GlcA to PmHS1 for chain elongation. Finally, dcbE encodes a homolog of E. coli kfiB,
and dcbB is a putative glycosyltransferase sharing homology with kfoG. Surprisingly, another heparosan synthase
(PmHS2) with similar glycosyltransferase domain organization and c. 70% identity to PmHS1 is located outside
of the CPS gene locus in the GAG-producing P. multocida type A, D, and F strains (DeAngelis & White, 2004).
UDP-glucose dehydrogenase –
EC 1.1.1.22 (kfiD )
2NADH + 2H+
UDP-GlcNAc
UDP-glucuronic acid
UDP
UDP
N -acetylglucosaminyltransferase –
EC 2.4.1.- (kfiA)
UDP-GlcA
glucuronosyltransferase –
EC 2.4.1.- (kfiC )
HEPAROSAN
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
Key:
Cofactors
Product
Fig. 8. Biosynthetic pathway for production
of K5 CPS, heparosan, in Escherichia coli K5.
FEMS Microbiol Rev 38 (2014) 660–697
679
Masquerading microbial pathogens
The biologic role of PmHS2 has not been characterized in
vivo, but it has been speculated that PmHS2 and the HA
and chondroitin synthases in type A and F strains, respectively, might be differentially regulated by environmental
conditions to allow variation in the type of GAG displayed on the cell surface (DeAngelis & White, 2004).
Owing to their broad substrate specificities compared
with PmHS1, purified PmHS2 and KfiA have been used
to synthesize novel unnatural polysaccharides from analogs of native UDP-sugar donors, and PmHS2 has also
been used to prepare heparosan for the chemoenzymatic
production of heparin (Liu et al., 2010; Li et al., 2013).
Heparosan in evasion of immune system
The structure of E. coli K5 CPS is identical to that of heparosan, which is the first polymeric intermediate during
the biosynthesis of heparin in the host (Navia et al., 1983).
In addition, the mature heparin chain also contains some
unsulfated and unepimerized [?4) b-D-GlcA (1?4) a-DGlcNAc (1?]n repeats identical to the K5 disaccharide
(Vann et al., 1981). Thus, the mimicry of the K5 CPS
structure makes it nonimmunogenic. As traditional CPS
typing relied on antigenicity, the capsule of E. coli K5 was
originally classified as K-nontypeable (Jann & Jann, 1987).
In part due to the difficulty of identifying the K5 antigen
and the frequency or extent in different infections, more
effective methods of typing were developed, including one
by Gupta et al. that utilized a specific phage to type K5 in
various infections (Kaijser & Jodal, 1984).
The capsules of E. coli K5 strains are most commonly
found in urinary tract infections (UTI; Sandberg et al.,
1988), such as pyelonephritis, cystitis, and asymptomatic
bacteriuria. Although K5 CPS has been found to be a
common E. coli capsule antigen in UTI infections, it is
also prevalent in strains causing sepsis (Kaijser & Jodal,
1984). In one of the studies using specific K5 phage typing, K5 was found in the 17.1% strains in the case of sepsis followed by 12.4% strains in the case of asymptomatic
bacteriuria (Kaijser & Jodal, 1984). Moreover, the K5 capsule has also been shown to promote the persistence of
E. coli in the rat large intestinal microbial communities
(Herias et al., 1997). The same study indicated that the
enhanced intestinal colonization of K5-encapsulated
strains allowed higher cell densities to be reached and secondarily led to increased translocation through the intestinal mucosa to mesenteric lymph nodes. Translocation is
thought to be a normal physiological process regulating
immunity to gut bacteria (Wells et al., 1988), but it can
also reach the blood stream causing sepsis and meningitis
(Lambert-Zechovsky et al., 1992). In the tissues, the capsule might further protect the organisms from phagocytosis and complement-mediated killing (Herias et al., 1997).
FEMS Microbiol Rev 38 (2014) 660–697
The heparosan CPS of the animal pathogen P. multocida
type D has also been demonstrated to be an important virulence factor for the organism. Acapsular variants of toxigenic P. multocida type D lose their virulence in a murine
model, and lesions caused by acapsular strains are less
severe than their encapsulated counterpart (Jacques et al.,
1993).
Hyaluronan
The unsulfated GAG known as HA exists in a wide-range
of mammalian tissues and as the primary constituent of
the CPS of several microorganisms, including the wellstudied bacteria S. pyogenes type A and C and P. multocida type A (other species possessing HA capsules are listed
in Table 1). Although the monosaccharide constituents of
this acidic polysaccharide are identical to heparosan, the
glycosidic linkages are distinct and lead to a slightly different repeating disaccharide structure. HA is noted as
one of the most hygroscopic molecules to exist in nature,
and it is estimated that hydrated HA contains a 1000-fold
mass of water compared with its own weight (Laurent &
Fraser, 1992). The high biocompatibility of HA stems
from its natural presence in many mammalian tissues,
and it is thus utilized in cosmetic and medical applications (Cimini et al., 2012), including tissue engineering
(Allison & Grande-Allen, 2006). HA has also long been
known as a regulator of cancer progression, and a highmolecular-mass HA unique to naked mole rats was
recently shown to mediate resistance to cancer, an unusual but famous property contributing to the species’
extraordinary longevity (Tian et al., 2013). Thus, commercial HA production is appealing for its use in a wide
range of applications. Industrial preparation of HA was
originally achieved by extraction of HA from animal
sources such as rooster combs, but there is interest in
moving toward more sustainable production practices
such as microbial fermentation (Boeriu et al., 2013). As
the existence of a bacterial HA synthase was first documented in pathogenic group A S. pyogenes (DeAngelis
et al., 1993a, b), HA production has been evaluated in
nonpathogenic Streptococci and recombinant strains (Yu
& Stephanopoulos, 2008; Yu et al., 2008; Liu et al., 2011),
and now, microbial HA has been fully commercialized
(DeAngelis, 2012).
Hyaluronan biosynthesis
Microbial production of HA has primarily been studied in
streptococcal species and P. multocida type A. Although
not all enzymes catalyzing biosynthetic steps toward HA
have been definitively proven, biosynthetic models have
been proposed based on known CPS genes in other
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
680
organisms (Liu et al., 2011; Cimini et al., 2012; Boeriu
et al., 2013). HA and heparosan share the same precursors
(UDP-GlcA and UDP-GlcNAc), so the metabolic models
for these CPSs are likely analogous. The similarity of early
biosynthetic steps for nucleotide-activated sugar precursors
across a wide range of bacteria also supports the assertion
that biosynthetic steps are conserved between heparosan
and HA strains. As UDP-glucose and UDP-GlcNAc are
important building blocks for many microbial glycans and
other glycoconjugates, it would be surprising to see significant interspecies variation in the anabolism of these critical
components. The obvious distinction between heparosan
and HA biosynthetic pathways is the glycosyltransferases
that polymerize the distinct CPSs. HA synthases from
Streptococci and P. multocida are bifunctional enzymes that
alternatively add GlcA and GlcNAc to the nascent polymer
(DeAngelis et al., 1993b, 1998; Kumari & Weigel, 1997;
Ward et al., 2001). As opposed to heparosan synthases that
form alternating a and b-linkages between monosaccharides, however, HA synthases only form b-linkages. A striking feature of HA synthases from different species is that
they polymerize in opposite directions; Class I HA synthases of Streptococci catalyze monosaccharide addition at the
reducing end of the chain, while Class II HA synthases in
P. multocida elongate the chain by addition to the nonreducing end (DeAngelis, 2012).
As discussed in previous sections, Regions 1 and 3 of
the P. multocida CPS gene cluster are presumably responsible for CPS transport through an ABC-transporterdependent pathway. Genes hyaB, hyaC, hyaD, and hyaE
in Region 2 of the P. multocida type A CPS locus are
involved in HA biosynthesis (Chung et al., 1998). Putative functions have been ascribed to P. multocida hyaB,
hyaC, and hyaE based upon their homology to kfoG,
kfiD/kfoF, and kfiB/kfoB, respectively, where HyaC is an
UDP-glucose dehydrogenase providing UDP-GlcA to HA
synthase, and HyaE could serve as a scaffold or structural
component. In fact, a P. multocida type A DhyaE mutant
was shown to be acapsular, suggesting that HyaE is critical for proper translocation of the HA CPS (Crouch
et al., 2012). In another study, an acapsular P. multocida
type A mutant was generated by deletion of hyaB, which
implicates HyaB as another key protein in CPS translocation (Steen et al., 2010). The hyaD gene encodes the HA
synthase PmHAS, an enzyme surprisingly sharing high
identity with the chondroitin synthase PmCS from
P. multocida type F (DeAngelis et al., 1998). Like most
other GAG synthases, PmHAS possesses two independent
glycosyltransferase domains that functions in a nonprocessive manner. Specifically, the N-terminus of PmHAS
possesses a GlcNAc-transferase domain, while the C-terminus possesses a GlcA-transferase domain (Jing &
DeAngelis, 2000; DeAngelis et al., 2003).
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
B.F. Cress et al.
Details regarding HA assembly in Gram-positive
Streptococci are lacking as well, but the HA biosynthetic
operon has been cloned from four species, including
Streptococcus pyogenes, Streptococcus uberis, Streptococcus
equisimilis, and Streptococcus zooepidemicus ssp. equi. This
operon possesses hasA (HA synthase) and hasB (UDPglucose dehydrogenase; homologous to hyaC in P. multocida type A) in all four species (Crater & van de Rijn,
1995). In all but S. uberis, hasC (UTP-glucose-1-P uridylyltransferase) is encoded downstream of hasB. In
S. uberis, hasC is located elsewhere in the genome (Ward
et al., 2001). The CPS operon in S. equisimilis and S. zooepidemicus also possesses hasD, a gene encoding the
bifunctional glucosamine-1-P N-acetyltransferase/UDPGlcNAc-1-P uridylyltransferase with homology to glmU
from Gram-negative bacteria. Finally, S. zooepidemicus
ssp. equi has an additional gene in the operon known as
hasE, which encodes phosphoglucoisomerase (Widner
et al., 2005). Phosphoglucoisomerase is essential for many
normal cellular processes, but with respect to CPS biogenesis, it could be important for balancing the intracellular abundance of the two UDP-sugar precursors to HA
(Chen et al., 2009; Prasad et al., 2010).
Genes encoded in the CPS operons of different species
might lead to more subtle variations in HA biogenesis
through the expression of dedicated enzymes that increase
the availability of UDP-sugar precursors to meet the
demands of CPS production. It is noteworthy that the
presence of such supplementary enzymes is inconsistent
between species producing identical CPS, as exemplified
by the extra copy of HasD in the CPS locus of S. equisimilis and S. zooepidemicus compared with S. uberis and
S. pyogenes (Blank et al., 2008). A possible explanation is
that dedicated biosynthetic enzymes might be required in
the metabolic background of one species to ensure sufficient intermediate metabolite availability, where, in a different species, sufficient precursor concentration exists
without these dedicated enzymes. It cannot be discounted
that the genetic difference is random; however, maintenance of function of these duplicate, CPS-dedicated
enzymes suggests that they confer an evolutionary advantage.
Hyaluronan in evasion of immune
system
Pasteurella multocida is an animal pathogen, mainly in
avian, bovine, and swine hosts, but can be transmitted to
humans through animal bites, particularly from cats and
dogs. Multiple studies have been performed that demonstrate the prevalence of the differing serotypes, based on
the different CPSs utilized, of P. multocida in healthy and
diseased organisms. In one study, 289 strains that were
FEMS Microbiol Rev 38 (2014) 660–697
Masquerading microbial pathogens
isolated from a variety of both healthy and ill animals
(including bovine, small ruminants, buffalo, swine, rabbits, dogs, cats, and poultry) were serotyped based on
PCR detection of the capsular biosynthesis genes capA, B,
D, E, and F. The study found that the HA capsule of type
A P. multocida was the most common among the isolates,
followed by type D (Ewers et al., 2006). A similar study
investigated the capsules found in porcine pneumonia
and atrophic rhinitis samples, and likewise found that A
was the most common, followed by D (Davies, 2003).
A 2012 study used multiplex PCR to determine the capsular genotype of isolates from 121 animals in Malaysia
and found that the capsular genotype was specific for
infections in different hosts, with capsular type A predominantly found in avian, rabbit, and porcine samples,
capsular type B found mostly in cattle and buffalo samples, capsular type D found mainly in goat samples, and
type F capsules found only in a cattle sample (Mohamad
et al., 2012). While the organism is most often associated
with disease in chickens and pigs, isolates have more
recently been studied from chimpanzees and humans. In
a study of isolates from the lungs of wild chimpanzees
affected by an outbreak of respiratory disease in 2004,
researchers found that all of the isolates had the biosynthesis genes required for the production of a HA capsule
(K€
ondgen et al., 2011). Another study, this time on 143
isolates of Pasteurella from humans, again found that type
A was the most common serotype identified, and it was
predominantly isolated from respiratory tissue, while isolates identified as type B, D, and F were found more
commonly than type A in soft tissue infections such as
bite wounds (Donnio et al., 2004).
The capsule of P. multocida serotype A is composed of
the GAG HA. Several studies have shown that this capsule
plays an important role in both virulence and evasion of
the host immune system. Studies on the capsule’s role in
virulence of P. multocida have shown that the HA capsule
enhances virulence and that inhibiting the production of
the capsule attenuates the pathogenicity. For instance,
one group showed that PBA930, a mutant P. multocida
type A strain deficient in capsule export, was attenuated
in mice when compared to the wild-type strain and that
lethality was restored to wild-type levels when the strain
was complemented with a plasmid encoding the genes
responsible for export (Chung et al., 2001). Another such
study showed that invasion of acapsular P. multocida
mutant cells into chicken embryo fibroblast cells was
decreased 12- to 16-fold when compared to the wild type
of the same strain (Al-haj Ali et al., 2004a, b). The same
study also showed that encapsulated strains were more
adhesive to the chicken embryo fibroblasts than the acapsular mutants. Interestingly, treatment with hyaluronidase
did not affect invasion or adhesion, while trypsin and
FEMS Microbiol Rev 38 (2014) 660–697
681
periodic acid treatments both inhibited invasion and
adhesion only in encapsulated strains, indicating that HA
may be playing a role in masking the receptors responsible for adhesion and invasion by these cells. Moreover,
another study showed that the thickness of the capsule
seems to also play a role in the virulence of P. multocida
infections in chickens, with increasing capsule thickness
correlating positively with virulence, with thinly capsulated strains being attenuated even at high doses (Borrathybay et al., 2003a).
Several studies have attempted to understand how the
HA capsule of P. multocida type A aids the bacteria in
evading the immune system of its host. Studies have
shown that this capsule aids in evading multiple facets of
the immune system, including phagocytic uptake, phagocytic killing, and the complement system. One such study
found that a mutant strain of P. multocida lacking the
genes required for capsular export was sensitive to killing
in 90% chicken serum, while encapsulated strains and
complemented mutant strains were not (Chung et al.,
2001). Interestingly, the group had previously determined
that the capsule of serotype B P. multocida, which has a
non-GAG capsule, composed of mannose, galactose, and
arabinose, did not confer resistance to complement in
chicken serum (Boyce et al., 2000b). This seems to indicate an important role in capsules composed of GAGs to
confer resistance to complement in serum.
In addition to resistance against complement activity in
serum, studies have also indicated that the HA capsule
plays a role in making the bacteria resistant to phagocytosis by macrophages. A 2004 study showed that immunization of mice with crude capsular extract (serotype A) did
not induce the production of antibodies against HA, but
did induce antibodies against a 39 kDa protein that is
only present in encapsulated strains of P. multocida. The
study also used immunoelectron microscopy to show that
the 39 kDa protein was localized at the capsule and that
mice that were immunized with the antibodies against
this protein were protected from at least 2 serotype
A strains of P. multocida (Al-haj Ali et al., 2004a, b). This
illustrates that one function of the capsule in promoting
virulence is to mask surface antigens on the bacterial cell
from the host immune system. Another study examined
the capsule’s role in virulence by comparing phagocytic
uptake of P. multocida cells with and without capsules
and found that encapsulated strains were resistant to
phagocytosis and that this resistance was lowered when
the capsules were removed enzymatically with hyaluronidase (Poermadjaja & Frost, 2000). Another study found
that encapsulated strains of P. multocida type A were less
hydrophobic than acapsular strains, which could be
responsible for inhibiting interactions between the cells
and hydrophobic components on the outer surface of
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
682
macrophages, and also found that treatment with hyaluronidase or mechanical shearing of the bacterial cells
significantly reduced the surface charge of the cells, which
could also play a role in interactions with macrophages
or other immune system components (Watt et al., 2003).
Together, the studies on the influence of the HA capsule
of P. multocida serotype A indicate that capsule enhances
virulence by evading the host immune system in a number of ways.
Streptococcus pyogenes, also commonly known as Group
A Streptococcus (GAS), is the causative agent of human
streptococcal pharyngitis, rheumatic fever, and soft tissue
infections including necrotizing fascitis. The capsule of
S. pyogenes is also composed of HA. Many studies have
been performed on GAS to elicit the importance and role
of the HA capsule in virulence. As in P. multocida, the
capsule of GAS has been shown to enhance virulence. An
interesting 2004 study showed that incidence of mucoid
(encapsulated) GAS isolates correlated temporally with
incidence of rheumatic fever (Veasy et al., 2004). One
group used mutations in the CsrRS regulation system to
show that increased capsule production correlated with
increased virulence (Engleberg et al., 2001). Another
study on GAS looked to determine the extent to which
the HA capsule contributed to virulence compared with
another important virulence factor in GAS, the M protein. Similar to the capsule of P. multocida type A, this
study found that the capsule of GAS played an important
role in resistance to phagocytosis in serum (Fillit et al.,
1986). Another study showed similar results, finding that
encapsulated GAS was able to grow in human blood,
while acapsular GAS was not, and that encapsulated GAS
was resistant to phagocytosis, while acapsular was not.
The same study found that the loss of the HA capsule
resulted in a 100-fold decrease of virulence in mice (Wessels et al., 1991).
In contrast to the immune-resistance roles in P. multocida, the HA capsule in GAS has been implicated in additional roles in virulence. One study using transmission
electron microscopy found that the binding of the GAS
capsule to skin epithelial CD-44 receptors induced cytoskeletal rearrangements that allowed the bacterial cells to
invade (Cywes & Wessels, 2001). However, an earlier
study found that the HA capsule of GAS did not correlate
with invasion into skin cells, but did find that the nonencapsulated GAS was significantly less virulent when it
invaded skin cells, causing fewer and less severe lesions
(Schrager et al., 1996). Another study, aiming to look at
the role of both the HA capsule and the M protein in a
baboon model, found that acapsular GAS persisted half as
long in the throat as encapsulated GAS and also indicated
a role for the capsule in enhancing microbial resistance to
antibody-mediated phagocytic killing (Ashbaugh et al.,
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
B.F. Cress et al.
2000). The studies on the importance of the HA capsule
in the virulence of Group A Streptococcus have shown
that its role is very similar to that of the HA capsule of
P. multocida. Its primary role seems to be to protect the
bacterial cell from host immune response, but it also has
additional roles in invasion of host cells.
Concluding remarks and future
perspectives
Owing to the diversity and abundance of distinct microbial virulence factors and their multifunctional, synergistic
properties, strictly decoupling the role of CPSs in pathogenicity from other contributing factors is a daunting and
risky task. The wealth of research devoted to bacterial
capsules has nevertheless expanded our understanding of
the mechanisms utilized by pathogens to persist in host
tissues and cavities without provoking severe immune
responses. In light of the concerning trend of pathogenic
strains evolving and acquiring drug resistance, however,
the scientific community should endeavor to identify vulnerabilities in this first line of defense for capsular pathogens and prioritize studies aimed at reducing virulence
through capsule manipulation or interference with capsular biosynthesis. Given the nature of this review, we
believe that a degree of speculation about possible studies
and therapeutic strategies is warranted in this perspectives
section.
Understanding virulence, pathogenicity,
and protective roles of capsules
Further studies will help to better understand the role of
the capsule in the pathogenicity of bacteria, as exemplified by the potential directions outlined in this section.
For instance, the presence or absence of the capsule
clearly impacts bacterial virulence, but little is known
about the effect of intermediate degrees of encapsulation.
Although CPS structure is likely the dominant virulence
determinant, the mass of CPS per cell, CPS chain length,
capsule density, and capsule thickness might be important
contributing factors to persistence against host immune
systems. A thick capsule of high molecular weight, nonimmunogenic polysaccharide might be expected to serve
as a more efficacious shield of cell surface components
than a tenuous coating composed of an identical polysaccharide of shorter chain length. Indeed, it should be
noted that strains of the same capsular type will express
different quantities or molecular weight of CPS (Hickey
et al., 2013), and it is clear that within many pathogenic
bacterial species, the more capsule expressed the more
virulent they are (Lee et al., 1991; Luong & Lee, 2002).
As there are other genetic differences between such
FEMS Microbiol Rev 38 (2014) 660–697
Masquerading microbial pathogens
wild-type strains, however, specifically implicating quantitative capsule expression as a virulence factor has not
been straightforward. Although studying the impact of
variable microbial capsule ‘coverage’ in animal infection
models is a difficult prospect, the relationship between
degree of coverage and pathogenicity is worth investigating because it could support or obviate the design of
drugs that facilitate clearance of encapsulated pathogens
through partial or complete removal of nonimmunogenic
CPSs. If even minimal capsule coverage is sufficient to
inhibit immune response, then a more appropriate drug
design strategy might target early stages of CPS biosynthesis to preclude capsule formation entirely.
The increasing prevalence of synthetic biology tools
could help resolve some intriguing questions about
immune response with respect to varying levels of encapsulation. Genetic mutations in past CPS virulence studies
have consistently been static, where the presence or
absence of capsule is set prior to inoculation by deleting
or heterologously expressing capsule biosynthesis genes.
One can envision engineering virulent wild-type strains
with dynamic transcriptional regulatory circuits capable
of modulating CPS production in response to environmental cues (Khalil & Collins, 2010; Chang et al., 2012a),
or even in an inducer-free, oscillatory manner that takes
advantage of mutually repressible genes (Elowitz & Leibler, 2000; Danino et al., 2010). Similarly, common metabolic engineering strategies such as overexpressing capsule
biosynthesis genes or downregulating and deleting genes
in competing metabolic pathways could be implemented
to create strains producing different quantities of CPS
(Yu & Stephanopoulos, 2008; Yu et al., 2008; Zhang
et al., 2012). After inoculating model animals with these
engineered strains, it is conceivable that the effect on
immunogenicity of transient variation in CPS coverage
could be monitored to understand critical coverage levels
and to measure rates of capsule assembly and degradation
in vivo. In any such studies, it will likely be important to
consider that differential capsule production might not
correlate with capsule coverage. For instance, if it can be
assumed that total mass of CPS attached to the outside of
a bacterium is intrinsically limited by surface area of the
outer leaflet or number of available attachment sites, then
one might ask, ‘Where would excess CPS localize after
this saturation point is reached?’ In strains engineered to
overexpress capsular biosynthetic machinery, it might be
expected that excess CPS would accumulate extracellularly
without leading to a corresponding augmentation of capsule thickness or density and without affecting immunogenicity. Alternatively, excess CPS might remain loosely
associated with the cell through noncovalent interactions
with the capsule, a scenario that could lead to capsule
thickening and attenuated host immune response. Related
FEMS Microbiol Rev 38 (2014) 660–697
683
questions that remain to be fully resolved are the following: can bacteria sense change in the quantity of CPS
attached to the outer membrane, and can such information actuate a change in CPS production levels? Alterations in bacterial metabolism upon capsule perturbation
could also be examined using metabolomic, transcriptomic, and proteomic studies to elucidate native capsule
regulatory mechanisms and other unknown players in
capsule assembly.
In contrast to modulating CPS production in vivo, an
alternative method for studying the relationship between
capsule coverage and immunogenicity would be to subject
capsular bacteria to CPS-specific lyases or glycosidases
that would remove the capsule postcolonization. For
instance, as carbohydrate-cleaving enzymes have been
shown to degrade bacterial capsules in vitro (Rimler et al.,
1995), it is conceivable that an infected animal could be
treated with these purified enzymes to strip the invading
pathogen of its capsule. Alternatively, pathogenic microorganisms could be engineered to secrete lyases or glycosidases to degrade their own CPS in situ, effectively
exposing cell surface antigenic determinants to the host
immune system on command from an external signal. In
a related example, the wild-type E. coli K5 strain naturally
produces and secretes a heparosan lyase that depolymerizes its own capsule (Legoux et al., 1996). Engineering the
secretion of enzymes that are not naturally secreted can
be challenging, but recent successes in this area suggest
that diverse types of recombinant proteins can be engineered for secretion in Gram-negative bacteria like E. coli
or Gram-positive bacteria like Bacillus subtilis or other
bacilli by fusing secretion peptide tags to one end of the
protein. Interested readers are directed to comprehensive
reviews on the subject (Simonen & Palva, 1993; Tjalsma
et al., 2004; Mergulhao et al., 2005). Prior to such studies, however, an additional question that should be
addressed is whether treatment with a lyase alters the
immunogenicity of CPS. The lyase from K5-specific coliphage creates an unnatural double bond in the externalfacing GlcA residue through a b-elimination mechanism
(H€anfling et al., 1996), which could presumably serve as
an antigenic ‘flag’ on the outer surface of the glycocalyx;
in contrast, treatment of K1 capsule with a sialidase (the
cognate polysialic acid glycosidase, or carbohydratehydrolyzing enzyme) would preserve the natural polysaccharide terminus without introduction of an unsaturated
bond. Thus, it is important to consider unintended
immunogenic consequences of the class of depolymerizing
enzyme utilized. To decouple the role of CPS structure
from other virulence factors, similar inducible circuits
could be designed to switch from expression of wild-type
CPS machinery to expression of biosynthetic enzymes for
the production of a different CPS structure, a scenario in
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
684
which the wild-type capsule would be progressively
supplanted by a capsule with dissimilar physical properties and antigens. In this manner, the confounding variables stemming from different virulence factors between
strains could be minimized when studying immune
response against distinct CPS structures.
Another valuable approach would be to develop genome-scale metabolic reconstructions of capsular bacteria
and study, in silico, environmental or media conditions
leading to differential CPS production level. Constraintsbased metabolic modeling techniques such as flux balance
analysis (Orth et al., 2010) and metabolite essentiality
analysis (MEA; Kim et al., 2007) excel at identifying nonintuitive strategies for modulating bacterial metabolism
toward a specific phenotype and have already been used to
study pathogenic bacteria and predict validated antibiotics
(Shen et al., 2010). By individually constraining production
of all metabolites to zero, one can probe the capacity of a
metabolic network to sustain a cellular phenotype (such as
maintaining biomass production/flux, the objective function of the optimization algorithm) when certain compounds are removed from metabolism. Metabolites that are
found to be essential for the phenotype of interest can be
derivatized to compete with the natural metabolite and hinder phenotype manifestation. In this manner, a novel antibiotic was rationally designed and shown to out-perform
an existing therapeutic for Vibrio vulnificus infection (Kim
et al., 2011). MEA could be applied in a similar manner to
genome-scale models of capsular pathogens, while using
CPS production as the objective function. As minimal subnetworks required to sustain the capsule are discovered in
simulations, gene deletions could be used to validate predictions in vivo. Analogs of essential CPS metabolites could
then be screened for CPS inhibition.
Although countless experiments could be contemplated,
the implication is clear: there is still much to learn about
the role of capsules in microbial pathogenesis, and creative strategies capitalizing on new genetic and computational tools should be devised to probe the limits of the
immune system in detecting and clearing exposed pathogens and to predict molecules capable of interfering with
capsule biosynthesis and transport. Exploration of the gap
between outright killing of pathogenic bacteria and simply exposing them to the immune system could lead to
development of therapeutic strategies for treating infection by multidrug-resistant strains, and it could also minimize society’s contribution to the alarming spread of
antibiotic resistance.
Potential therapeutic approaches
Therapeutic strategies that replace or supplement antibiotic treatment by capitalizing on capsule susceptibility
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
B.F. Cress et al.
will likely depend upon the type of CPS expressed by the
invading pathogen, and thus, development of rapid and
inexpensive diagnostic tools to guide treatment options
will be a critical component of any therapeutic strategy.
Serological characterization of surface antigens is hindered by the inability to generate, with high specificity,
antibodies against important CPS structures that are identical to human glycans. Although MLST analyses and
PCR based techniques guided by conserved capsule flanking regions are the current gold standard for molecular
typing, these methods are limited due to the requirement
of equipment that is not ubiquitous. Furthermore, molecular typing could lead to false positives due to expectations of CPS production that might not manifest due to
the presence of mutations or additional CPS-modifying
enzymes located outside of the assayed gene loci. Rapid
CPS diagnostics should be cheap, deployable to any location in the world, and should exhibit very high specificity
for the target molecule to limit false positives.
One technology that satisfies these constraints is known
as a molecular beacon, or a nucleic acid aptamer that has
been engineered to emit a fluorometric or colorimetric
signal upon binding of target molecules (Raj & van
Oudenaarden, 2009). Aptamers are nucleic acid oligomers
that have been selected from large libraries of random
sequences due to their affinity and selectivity for a target
molecule (Voigt, 2006). Highly specific aptamer affinity
to a target molecule is easily attained by performing negative selection screens against structurally related molecules
to remove cross-reacting oligonucleotides from the candidate aptamer pool. Aptamers with dissociation constants
in the low nanomolar range have been generated against
carbohydrates (Sun et al., 2010) and even recently against
the PSA from the capsule of E. coli K1 (Cho et al., 2013).
Target binding is associated with conformation change in
the oligonucleotide; hence, molecular beacons can be
readily designed with covalently bound fluorophore and
quencher molecules that remain in close proximity in the
unbound state to mask fluorescence but separate enough
to unveil the fluorescent moiety when bound to the target
(Tombelli et al., 2005). Addition of molecular beacon to
a sample containing the cognate CPS would enable fluorescence but would require a high intensity light source
for excitation and detection. Alternatively, aptamer
sequences have been fused with ribozymes to, upon binding of target molecule, actuate production of a colored
compound by reaction with other reagents in solution
(Vinkenborg et al., 2011; Tang et al., 2012). Diagnosis of
CPS structure with molecular beacons would be rapid,
cheap, and easily performed by any healthcare worker.
Subsequent to diagnosis of the predominant CPSs, targeted therapeutic strategies can be implemented. One
strategy for treatment of intestinal infections might
FEMS Microbiol Rev 38 (2014) 660–697
685
Masquerading microbial pathogens
involve ingestion of a probiotic strain that has been previously characterized to exclude or inhibit pathogens
(Fig. 9a; Lebeer et al., 2010). The term ‘probiotics’
describes microorganisms that confer health benefits to
the host, particularly with respect to human health
(Marco et al., 2006). For instance, the probiotic strain
E. coli Nissle 1917 has been shown to outcompete the
encapsulated uropathogenic E. coli (UPEC) strain CFT073
in several growth conditions, including growth in urine
(Hancock et al., 2010), and it exhibits bactericidal activity
against many other pathogenic microorganisms as well
(Storm et al., 2011). Prophylactic administration of Nissle
1917 in a porcine model was even shown to abolish
secretory diarrhea upon challenge with an ETEC strain
(Schroeder et al., 2006). In addition to the ability of Nissle 1917 to outcompete pathogens, it has been shown in
another experiment using a porcine model to persist in
the gut for up to a month after inoculation (Barth et al.,
2009). Nissle 1917 is of particular interest in this review
because of its nonimmunogenic heparosan capsule, which
might confer an advantage over other probiotics in outcompeting extraintestinal pathogens in, for example, the
urinary tract. Furthermore, the ease of genetic manipulation of E. coli compared with other bacteria could make
Nissle 1917 a model probiotic target for more
complicated engineering strategies. The most common
probiotics in use today are, however, members of the Lactobacillus and Bifidobacterium genera, which are utilized
primarily in the GI tract. In addition, previous work has
demonstrated the feasibility of engineering probiotic bacteria for vaccine delivery, as exemplified by a study in
which nasal administration of Lactococcus lactis, recombinantly expressing pneumococcal protective protein on its
surface, induced protection against Streptococcus pneumoniae infection in young mice (Vinti~
ni et al., 2010).
A related but speculative therapeutic strategy would be
engineering probiotic strains to detect pathogenic signals
and secrete pathogen-specific CPS-depolymerizing
enzymes, effectively presenting the surface of the pathogen to the host immune system in a strategy that might
complement any other positive effects already conferred
by the probiotic strain (Fig. 9c). This strategy would
require engineering a sensor capable of controlling
expression and secretion of a CPS-degrading enzyme.
One candidate is known as a riboregulator, an RNA
sequence capable of binding a ligand through an aptamer
domain, which induces a conformational change sequestering an antisense domain and allowing expression of
(a)
(b)
(c)
(d)
Fig. 9. Strategies to combat capsule-bearing pathogens. Probiotic or engineered strains are designated in blue, and pathogenic strains are
colored red with or without an orange capsule. (a) Ingestion of wild-type probiotic or engineered strains that outcompete pathogenic bacteria.
(b) Small-molecule inhibitors of CPS biosynthetic enzymes (upper) or CPS translocation proteins (lower). Triangles represent small-molecule
inhibitors; purple circular (‘Pac-Man’) symbol represents polysaccharide glycosyltransferase. (c) Treatment with probiotic bacteria engineered to
secrete lyases or glycosidases (drawn as scissors) with activity against pathogen’s CPS (upper) or treatment with purified enzyme (lower).
(d) Treatment with natural or engineered bacteriophage to lyse bacteria bearing specific CPS type.
FEMS Microbiol Rev 38 (2014) 660–697
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
686
the cognate mRNA. Riboregulators have been used to control translation of proteins in response to various ligands
as discussed elsewhere (Khalil & Collins, 2010); thus, it is
expected that incorporation of an RNA aptamer domain
capable of binding a specific CPS could be incorporated
into a riboregulator to control expression of a secretiontagged, CPS-depolymerizing lyase or glycosidase. An alternative to engineering enzyme-secreting probiotic strains
would be to simply purify recombinantly expressed CPScleaving enzymes from fermentations of genetically tractable, secretion-capable production strains and to administer
the purified enzyme to the patient. In one study illustrating the potential of this strategy, intraperitoneal injection
of an anti-K1 glycosidase (endosialidase E from a bacteriophage) to rat pups with previously induced blood-borne
E. coli K1 infection significantly reduced mortality and
also showed prophylactic efficacy (Mushtaq et al., 2004).
The glycosidase treatment was shown not reduce the viability of the pathogen, but rather to expose the bacteria to
complement-mediated killing; bacteremia typically subsided within 24 h postinjection and prevented death in
most cases. Many studies have also demonstrated the
promise of alginate lyase therapeutics for degrading Pseudomonas aeruginosa biofilms in the airways of cystic fibrosis patients, including a recent investigation of alginate
lyase-PEG conjugates for degradation of P. aeruginosa biofilms on abiotic surfaces (Lamppa et al., 2011).
Phage therapy is another approach that should be considered for its potential to combat pathogenic bacteria
possessing nonimmunogenic capsules (Fig. 9d). Interest in
the use of bacteriophages, or viruses that infect bacteria,
declined in the West after the advent of antibiotics, but
the alarming trend of drug-resistant bacterial infections
has provoked reconsideration throughout the last decade
of phage therapy as a strategy to replace or potentiate antibiotic treatment. Despite the decreasing investigation of
phage therapy in Europe and the Americas after the commercialization of antibiotics, much work devoted to this
topic was continued in states of the former Soviet Union
and Poland throughout the twentieth century, including
the use of bacteriophage to treat human diseases (O’Flaherty et al., 2009). An excellent review details the successful
application of phage therapy for a wide range of infections
and addresses the safety of phages from a medical standpoint (Sulakvelidze et al., 2001). A practical example of
phage treatment includes the U.S. Food and Drug Administration’s approval in 2006 of the use of a phage cocktail
to prevent adulteration of ready-to-eat meat products by
Listeria monocytogenes (Shuren, 2006). Furthermore,
promising recent work has demonstrated the ability to
engineer bacteriophages like T7 to degrade infectious biofilms (Azeredo & Sutherland, 2008) by expressing CPSspecific (Scholl et al., 2005) or EPS-specific (Lu & Collins,
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
B.F. Cress et al.
2007) depolymerizing enzymes on their surface or tail
spike, or by expressing these enzymes intracellularly during infection for subsequent release and targeting of neighboring bacterial cells after lysis. One strategy that phages
have naturally evolved is the ability to ‘dig’ for cell surface
receptors that have been masked by cell surface biomolecules, such as CPS, by depolymerizing the coating. This is
exemplified by K1- and K5-specific coliphages (Stummeyer
et al., 2004; Thompson et al., 2010) that express evolved
tail spike proteins capable of CPS-specific degradation. To
date, various bacteriophages have been shown to possess
CPS-degrading enzymes specific for heparosan (Thompson
et al., 2010), polysialic acid (Stummeyer et al., 2004), and
hyaluronan (Baker et al., 2002), but no bacteriophages are
known to possess chondroitin lyases. However, bacterial
enzymes have been successfully expressed by bacteriophage, so it could be valuable to explore bacteriophage
surface or tail spike expression of bacterial chondroitinases
to target and depolymerize the chondroitin capsules of
certain pathogenic P. multocida and E. coli strains. Taken
together, these studies not only highlight the potential to
utilize highly specific phages as antibacterial agents against
encapsulated bacteria, but they suggest that it should be
possible to engineer bacteriophages to specifically target
and lyse pathogenic microorganisms possessing nonimmunogenic capsules.
Finally, small-molecule therapeutics could be used to
interfere with CPS biosynthesis, CPS translocation, or
even CPS attachment to the outer leaflet of the outer
membrane (Fig. 9b). In a recent publication, Seed et al.
developed a high-throughput screening method to search
large chemical libraries for capsule inhibitors (Goller &
Seed, 2010). Using a CPS-dependent bacteriophage (that
requires surface-exposed capsule for adsorption and subsequent ejection of nucleic acid from phage into host) as
an indicator for the presence of intact capsule, bacteria
that had been incubated with library compounds were
evaluated for capsule production. One compound,
dubbed ‘C7’, inhibited capsule formation in UTI89, a
UPEC K1 strain. In a fascinating turn of events, C7 also
inhibited capsule formation in UPEC K5 strain DS17.
Due to the finding that assembly of two different CPS
structures was inhibited by the same compound, coupled
with data showing insignificant accumulation of intracellular or extracellular CPS, it was hypothesized that C7
inhibits an early stage of CPS biogenesis in all Group 2
capsular E. coli. This study exemplifies the approaches
that could lead to incredibly valuable therapeutics against
infectious pathogens. Very recently, another study demonstrated the use of rationally designed glycomimetics to
block export of an E. coli CPS by ‘plugging’ Wza, a
pore-like translocon protein in the Wzy-dependent transport pathway (Kong et al., 2013). It is our hope that
FEMS Microbiol Rev 38 (2014) 660–697
Masquerading microbial pathogens
aforementioned strategies will contribute to a better
understanding of capsule biosynthesis in a metabolic context and inspire discussion leading to CPS-targeting therapeutic and prophylactic solutions to a host of serious
diseases caused by masquerading microbial pathogens.
Acknowledgements
We would like to thank all of our coworkers and collaborators, as well as the funding agencies that make this
work possible. B. F. Cress, J. A. Englaender, and W. He
are partially supported by graduate fellowships from
Rennselaer Polytechnic Institute. Research in the Linhardt
laboratory on the subject of this review is supported by
NIH Grants HL62244, HL094463, and HL096972.
References
Adlam C, Knights JM, Mugridge A, Williams JM & Lindon JC
(1987) Production of colominic acid by Pasteurella
haemolytica serotype A2 organisms. FEMS Microbiol Lett 42:
23–25.
Al-haj Ali H, Sawada T, Hatakeyama H, Katayama Y, Ohtsuki
N & Itoh O (2004a) Invasion of chicken embryo fibroblast
cells by avian Pasteurella multocida. Vet Microbiol 104:
55–62.
Al-haj Ali H, Sawada T, Hatakeyama H, Ohtsuki N & Itoh O
(2004b) Characterization of a 39kDa capsular protein of
avian Pasteurella multocida using monoclonal antibodies.
Vet Microbiol 100: 43–53.
Allison DD & Grande-Allen KJ (2006) Review. Hyaluronan: a
powerful tissue engineering tool. Tissue Eng 12: 2131–2140.
Almeida RA & Oliver SP (1993) Antiphagocytic effect of the
capsule of Streptococcus uberis. Zentralbl Veterinarmed B 40:
707–714.
Almeida A, Albuquerque P, Araujo R, Ribeiro N & Tavares F
(2013) Detection and discrimination of common bovine
mastitis-causing streptococci. Vet Microbiol 164: 370–377.
Andreishcheva EN & Vann WF (2006) Escherichia coli BL21
(DE3) chromosome contains a group II capsular gene
cluster. Gene 384: 113–119.
Annunziato PW, Wright LF, Vann WF & Silver RP (1995)
Nucleotide sequence and genetic analysis of the neuD and
neuB genes in region 2 of the polysialic acid gene cluster of
Escherichia coli K1. J Bacteriol 177: 312–319.
Anzai T, Timoney JF, Kuwamoto Y, Fujita Y, Wada R & Inoue
T (1999) In vivo pathogenicity and resistance to
phagocytosis of Streptococcus equi strains with different
levels of capsule expression. Vet Microbiol 67: 277–286.
Ashbaugh CD, Moser TJ, Shearer MH, White GL, Kennedy
RC & Wessels MR (2000) Bacterial determinants of
persistent throat colonization and the associated immune
response in a primate model of human group A
streptococcal pharyngeal infection. Cell Microbiol 2:
283–292.
FEMS Microbiol Rev 38 (2014) 660–697
687
Avci FY & Kasper DL (2010) How bacterial carbohydrates
influence the adaptive immune system. Annu Rev Immunol
28: 107–130.
Azeredo J & Sutherland IW (2008) The use of phages for the
removal of infectious biofilms. Curr Pharm Biotechnol 9:
261–266.
Baker J, Dong S & Pritchard D (2002) The hyaluronan lyase of
Streptococcus pyogenes bacteriophage H4489A. Biochem J
365: 317–322.
Barrett B, Ebah L & Roberts IS (2002) Genomic structure of
capsular determinants. Curr Top Microbiol 264: 137–155.
Barth S, Duncker S, Hempe J, Breves G, Baljer G & Bauerfeind
R (2009) Escherichia coli Nissle 1917 for probiotic use in
piglets: evidence for intestinal colonization. J Appl Microbiol
107: 1697–1710.
Bendaoud M, Vinogradov E, Balashova NV, Kadouri DE,
Kachlany SC & Kaplan JB (2011) Broad-spectrum biofilm
inhibition by Kingella kingae exopolysaccharide. J Bacteriol
193: 3879–3886.
Bitter-Suermann D, Goergen I & Frosch M (1986) Monoclonal
antibodies to weak immunogenic Escherichia coli and
meningococcal capsular polysaccharides.
Protein-Carbohydrate Interactions in Biological Systems: The
Molecular Biology of Microbial Pathogenicity (Lark DL, ed.),
pp. 395–396. Academic Press, London.
Blank LM, Hugenholtz P & Nielsen LK (2008) Evolution of
the hyaluronic acid synthesis (has) operon in Streptococcus
zooepidemicus and other pathogenic streptococci. J Mol Evol
67: 13–22.
Bliss JM, Garon CF & Silver RP (1996) Polysialic acid export
in Escherichia coli K1: the role of KpsT, the ATP-binding
component of an ABC transporter, in chain translocation.
Glycobiology 6: 445–452.
Boeriu CG, Springer J, Kooy FK, van den Broek LAM &
Eggink G (2013) Production methods for hyaluronan. Int J
Carbohydr Chem 2013: 1–14.
Born J, Klaus J, Assmann KJ, Lindahl U & Berden JH (1996)
N-Acetylated domains in heparan sulfates revealed by a
monoclonal antibody against the Escherichia coli K5
capsular polysaccharide. Distribution of the cognate
epitope in normal human kidney and transplant kidney
with chronic vascular rejection. J Biol Chem 271:
22802–22809.
Borrathybay E, Sawada T, Kataoka Y, Okiyama E, Kawamoto
E & Amao H (2003a) Capsule thickness and amounts of a
39kDa capsular protein of avian Pasteurella multocida type
A strains correlate with their pathogenicity for chickens. Vet
Microbiol 97: 215–227.
Borrathybay E, Sawada T, Kataoka Y, Ohtsu N, Takagi M,
Nakamura S & Kawamoto E (2003b) A 39kDa protein
mediates adhesion of avian Pasteurella multocida to chicken
embryo fibroblast cells. Vet Microbiol 97: 229–243.
Bortolussi R, Ferrieri P, Bj€
orksten B & Quie PG (1979)
Capsular K1 polysaccharide of Escherichia coli: relationship
to virulence in newborn rats and resistance to phagocytosis.
Infect Immun 25: 293–298.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
688
Bøvre K, Bryn K, Closs O, Hagen N & Frøholm LO (1983)
Surface polysaccharide of Moraxella non-liquefaciens
identical to Neisseria meningitidis group B capsular
polysaccharide. A chemical and immunological
investigation. NIPH Ann 6: 65–73.
Boyce JD, Chung JY & Adler B (2000a) Genetic organisation
of the capsule biosynthetic locus of Pasteurella multocida
M1404 (B:2). Vet Microbiol 72: 121–134.
Boyce JD, Chung JY & Adler B (2000b) Pasteurella multocida
capsule: composition, function and genetics. J Biotechnol 83:
153–160.
Boyce JD, Harper M, Wilkie IW & Adler B (2010) Pasteurella.
Pathogenesis of Bacterial Infections in Animals, 4th edn (Gyles
CL, Prescott J, Songer G & Thoen CO, eds), pp. 325–346.
Wiley-Blackwell, DOI: 10.1002/9780470958209.ch17.
Bronner D, Sieberth V, Pazzani C, Smith A, Boulnois G, Roberts
I, Jann B & Jann K (1993) Synthesis of the K5 (group II)
capsular polysaccharide in transport-deficient recombinant
Escherichia coli. FEMS Microbiol Lett 113: 279–284.
Brooks HJ, O’Grady F, McSherry MA & Cattell WR (1980)
Uropathogenic properties of Escherichia coli in recurrent
urinary-tract infection. J Med Microbiol 13: 57–68.
Bushell SR, Mainprize IL, Wear MA, Lou H, Whitfield C &
Naismith JH (2013) Wzi is an outer membrane lectin that
underpins Group 1 capsule assembly in Escherichia coli.
Structure 21: 844–853.
Byarugaba DK, Minga UM, Gwakisa PS,
Katunguka-Rwakishaya E, Bisgaard M & Olsen JE (2007)
Virulence characterization of Avibacterium paragallinarum
isolates from Uganda. Avian Pathol 36: 35–42.
Calvinho LF, Almeida RA & Oliver SP (1998) Potential
virulence factors of Streptococcus dysgalactiae associated with
bovine mastitis. Vet Microbiol 61: 93–110.
Capila I & Linhardt RJ (2002) Heparin-protein interactions.
Angew Chem Int Edit 41: 391–412.
Chang RCC, Chiu K, Ho YS & So KF (2009) Modulation of
neuroimmune responses on glia in the central nervous
system: implication in therapeutic intervention against
neuroinflammation. Cell Mol Immunol 6: 317–326.
Chang AL, Wolf JJ & Smolke CD (2012a) Synthetic RNA
switches as a tool for temporal and spatial control over gene
expression. Curr Opin Biotechnol 23: 679–688.
Chang Y, Yang B, Zhao X & Linhardt RJ (2012b) Analysis of
glycosaminoglycan-derived disaccharides by capillary
electrophoresis using laser-induced fluorescence detection.
Anal Biochem 427: 91–98.
Chen X & Varki A (2010) Advances in the biology and
chemistry of sialic acids. ACS Chem Biol 5: 163–176.
Chen SL, Hung CS, Xu J et al. (2006) Identification of genes
subject to positive selection in uropathogenic strains of
Escherichia coli: a comparative genomics approach. P Natl
Acad Sci USA 103: 5977–5982.
Chen WY, Marcellin E, Hung J & Nielsen LK (2009)
Hyaluronan molecular weight is controlled by
UDP-N-acetylglucosamine concentration in Streptococcus
zooepidemicus. J Biol Chem 284: 18007–18014.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
B.F. Cress et al.
Cho S, Lee BR, Cho BK, Kim JH & Kim BG (2013) In vitro
selection of sialic acid specific RNA aptamer and its
application to the rapid sensing of sialic acid modified
sugars. Biotechnol Bioeng 110: 905–913.
Chung JY, Zhang Y & Adler B (1998) The capsule biosynthetic
locus of Pasteurella multocida A:1. FEMS Microbiol Lett 166:
289–296.
Chung JY, Wilkie I, Boyce JD, Townsend KM, Frost AJ,
Ghoddusi M & Adler B (2001) Role of capsule in the
pathogenesis of fowl cholera caused by Pasteurella multocida
serogroup A. Infect Immun 69: 2487–2492.
Cieslewicz M & Vimr E (1996) Thermoregulation of kpsF, the
first region 1 gene in the kps locus for polysialic acid
biosynthesis in Escherichia coli K1. J Bacteriol 178: 3212–3220.
Cimini D, De Rosa M & Schiraldi C (2012) Production of
glucuronic acid-based polysaccharides by microbial
fermentation for biomedical applications. Biotechnol J 7:
237–250.
Cimini D, De Rosa M, Carlino E, Ruggiero A & Schiraldi C
(2013) Homologous overexpression of rfaH in E. coli K4
improves the production of chondroitin-like capsular
polysaccharide. Microb Cell Fact 12: 1–12.
Cobb BA, Wang Q, Tzianabos AO & Kasper DL (2004)
Polysaccharide processing and presentation by the MHCII
pathway. Cell 117: 677–687.
Comstock LE & Kasper DL (2006) Bacterial glycans: key
mediators of diverse host immune responses. Cell 126: 847–
850.
Costerton JW, Cheng KJ, Geesey GG, Ladd TI, Nickel JC,
Dasgupta M & Marrie TJ (1987) Bacterial biofilms in nature
and disease. Annu Rev Microbiol 41: 435–464.
Crater DL & van de Rijn I (1995) Hyaluronic acid synthesis
operon (has) expression in group A streptococci. J Biol
Chem 270: 18452–18458.
Cress BF, Linhardt RJ & Koffas MAG (2013a) Draft genome
sequence of Escherichia coli strain Nissle 1917 (Serovar O6:
K5:H1). Genome Announc 1: 1–2.
Cress BF, Greene ZR, Linhardt RJ & Koffas MAG (2013b)
Draft genome sequence of Escherichia coli strain ATCC
23502 (Serovar O5:K4:H4). Genome Announc 1: 1–2.
Cress BF, Greene ZR, Linhardt RJ & Koffas MAG (2013c)
Draft genome sequence of Escherichia coli strain ATCC
23506 (Serovar O10:K5:H4). Genome Announc 1: 1–2.
Cross AS (1990) The biologic significance of bacterial
encapsulation. Curr Top Microbiol 150: 87–95.
Crouch CF, LaFleur R, Ramage C, Reddick D, Murray J,
Donachie W & Francis MJ (2012) Cross protection of a
Mannheimia haemolytica A1 Lkt-/Pasteurella multocida
DhyaE bovine respiratory disease vaccine against
experimental challenge with Mannheimia haemolytica A6 in
calves. Vaccine 30: 2320–2328.
Cunningham RK, S€
oderstr€
om TO, Gillman CF & Van Oss CJ
(1975) Phagocytosis as a surface phenomenon. V. Contact
angles and phagocytosis of rough and smooth strains of
Salmonella typhimurium, and the influence of specific
antiserum. Immunol Commun 4: 429–442.
FEMS Microbiol Rev 38 (2014) 660–697
Masquerading microbial pathogens
Cywes C & Wessels MR (2001) Group A Streptococcus tissue
invasion by CD44-mediated cell signalling. Nature 414: 648–
652.
Daines DA & Silver RP (2000) Evidence for multimerization of
neu proteins involved in polysialic acid synthesis in
Escherichia coli K1 using improved LexA-based vectors.
J Bacteriol 182: 5267–5270.
Danino T, Mondrag
on-Palomino O, Tsimring L & Hasty J
(2010) A synchronized quorum of genetic clocks. Nature
463: 326–330.
Dasgupta S & Kasper DL (2010) Novel tools for modulating
immune responses in the host-polysaccharides from
the capsule of commensal bacteria. Adv Immunol 106:
61–91.
Davies RL (2003) Characterization and comparison of
Pasteurella multocida strains associated with porcine
pneumonia and atrophic rhinitis. J Med Microbiol 52:
59–67.
DeAngelis PL (1999) Hyaluronan synthases: fascinating
glycosyltransferases from vertebrates, bacterial pathogens,
and algal viruses. Cell Mol Life Sci 56: 670–682.
DeAngelis PL (2002a) Evolution of glycosaminoglycans and
their glycosyltransferases: Implications for the extracellular
matrices of animals and the capsules of pathogenic bacteria.
Anat Rec 268: 317–326.
DeAngelis PL (2002b) Microbial glycosaminoglycan
glycosyltransferases. Glycobiology 12: 9R–16R.
DeAngelis PL (2012) Glycosaminoglycan polysaccharide
biosynthesis and production: today and tomorrow. Appl
Microbiol Biotechnol 94: 295–305.
DeAngelis PL & Padgett-McCue AJ (2000) Identification and
molecular cloning of a chondroitin synthase from
Pasteurella multocida type F. J Biol Chem 275: 24124–24129.
DeAngelis PL & White CL (2002) Identification and molecular
cloning of a heparosan synthase from Pasteurella multocida
type D. J Biol Chem 277: 7209–7213.
DeAngelis PL & White CL (2004) Identification of a distinct,
cryptic heparosan synthase from Pasteurella multocida types
A, D, and F. J Bacteriol 186: 8529–8532.
DeAngelis PL, Papaconstantinou J & Weigel PH (1993a)
Isolation of a Streptococcus pyogenes gene locus that directs
hyaluronan biosynthesis in acapsular mutants and in
heterologous bacteria. J Biol Chem 268: 14568–14571.
DeAngelis PL, Papaconstantinou J & Weigel PH (1993b)
Molecular cloning, identification, and sequence of the
hyaluronan synthase gene from group A Streptococcus
pyogenes. J Biol Chem 268: 19181–19184.
DeAngelis PL, Jing W, Drake RR & Achyuthan AM (1998)
Identification and molecular cloning of a unique hyaluronan
synthase from Pasteurella multocida. J Biol Chem 273: 8454–
8458.
DeAngelis PL, Gunay NS, Toida T, Mao W & Linhardt RJ
(2002) Identification of the capsular polysaccharides of
Type D and F Pasteurella multocida as unmodified heparin
and chondroitin, respectively. Carbohydr Res 337:
1547–1552.
FEMS Microbiol Rev 38 (2014) 660–697
689
DeAngelis PL, Oatman LC & Gay DF (2003) Rapid
chemoenzymatic synthesis of monodisperse hyaluronan
oligosaccharides with immobilized enzyme reactors. J Biol
Chem 278: 35199–35203.
Deszo EL, Steenbergen SM, Freedberg DI & Vimr ER (2005)
Escherichia coli K1 polysialic acid O-acetyltransferase gene,
neuO, and the mechanism of capsule form variation
involving a mobile contingency locus. P Natl Acad Sci USA
102: 5564–5569.
Devi SJ, Schneerson R, Egan W, Vann WF, Robbins JB &
Shiloach J (1991) Identity between polysaccharide antigens
of Moraxella nonliquefaciens, group B Neisseria meningitidis,
and Escherichia coli K1 (non-O acetylated). Infect Immun 59:
732–736.
Donnio PY, Lerestif-Gautier AL & Avril JL (2004)
Characterization of Pasteurella spp. strains isolated from
human infections. J Comp Pathol 130: 137–142.
Dougherty BA & van de Rijn I (1992) Molecular
characterization of a locus required for hyaluronic acid
capsule production in group A streptococci. J Exp Med 175:
1291–1299.
Durso LM, Bono JL & Keen JE (2005) Molecular serotyping of
Escherichia coli O26:H11. Appl Environ Microbiol 71: 4941–
4944.
Edwards U & Frosch M (1992) Sequence and functional
analysis of the cloned Neisseria meningitidis CMP-NeuNAc
synthetase. FEMS Microbiol Lett 75: 161–166.
Edwards MS, Kasper DL, Jennings HJ, Baker CJ &
Nicholson-Weller A (1982) Capsular sialic acid prevents
activation of the alternative complement pathway by
type III, group B streptococci. J Immunol 128:
1278–1283.
Elowitz MB & Leibler S (2000) A synthetic oscillatory network
of transcriptional regulators. Nature 403: 335–338.
Engleberg NC, Heath A, Miller A, Rivera C & DiRita VJ
(2001) Spontaneous mutations in the CsrRS
two-component regulatory system of Streptococcus pyogenes
result in enhanced virulence in a murine model of skin
and soft tissue infection. J Infect Dis 183: 1043–1054.
Erbel PJA, Barr K, Gao N, Gerwig GJ, Rick PD & Gardner KH
(2003) Identification and biosynthesis of cyclic
enterobacterial common antigen in Escherichia coli.
J Bacteriol 185: 1995–2004.
Esko JD (1999) Proteoglycans and glycosaminoglycans.
Essentials of Glycobiology (Varki A, Cummings R, Esko J,
Freeze H, Hart G & Marth J, eds). Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY.
Esko JD, Doering TL & Raetz CR (2009) Eubacteria and
Archaea. Essentials of Glycobiology, 2nd edn (Varki A,
Cummings RD, Esko JD, Freeze HH, Stanley P,
Bertozzi CR, Hart GW & Etzler ME, eds). Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY.
Ewers C, L€
ubke-Becker A, Bethe A, Kiebling S, Filter M &
Wieler LH (2006) Virulence genotype of Pasteurella
multocida strains isolated from different hosts with various
disease status. Vet Microbiol 114: 304–317.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
690
Fillit HM, McCarty M & Blake M (1986) Induction of
antibodies to hyaluronic acid by immunization of rabbits
with encapsulated Streptococci. J Exp Med 164: 762–776.
Finke A, Bronner D, Nikolaev AV, Jann B & Jann K (1991)
Biosynthesis of the Escherichia coli K5 polysaccharide, a
representative of group II capsular polysaccharides:
polymerization in vitro and characterization of the product.
J Bacteriol 173: 4088–4094.
Finne J (1982) Occurrence of unique polysialosyl carbohydrate
units in glycoproteins of developing brain. J Biol Chem 257:
11966–11970.
Finne J, Leinonen M & M€akel€a PH (1983) Antigenic
similarities between brain components and bacteria causing
meningitis. Implications for vaccine development and
pathogenesis. Lancet 2: 355–357.
Fregolino E, Ivanova R, Lanzetta R, Molinaro A, Parrilli M,
Paunova-Krasteva T, Stoitsova SR & De Castro C (2012)
Occurrence and structure of cyclic enterobacterial common
antigen in Escherichia coli O157:H . Carbohydr Res 363: 29–32.
Frosch M & M€
uller A (1993) Phospholipid substitution of
capsular polysaccharides and mechanisms of capsule
formation in Neisseria meningitidis. Mol Microbiol 8: 483–
493.
Frosch M, G€
orgen I, Boulnois GJ, Timmis KN &
Bitter-Suermann D (1985) NZB mouse system for
production of monoclonal antibodies to weak bacterial
antigens: isolation of an IgG antibody to the polysaccharide
capsules of Escherichia coli K1 and group B meningococci.
P Natl Acad Sci USA 82: 1194–1198.
Frosch M, Edwards U, Bousset K, Krausse B & Weisgerber C
(1991) Evidence for a common molecular origin of the
capsule gene loci in gram-negative bacteria expressing group
II capsular polysaccharides. Mol Microbiol 5: 1251–1263.
Gagneux P & Varki A (1999) Evolutionary considerations in
relating oligosaccharide diversity to biological function.
Glycobiology 9: 747–755.
Gallagher JT (1989) The extended family of proteoglycans:
social residents of the pericellular zone. Curr Opin Cell Biol
1: 1201–1218.
Ganguli S, Zapata G, Wallis T, Reid C, Boulnois G, Vann WF
& Roberts IS (1994) Molecular cloning and analysis of genes
for sialic acid synthesis in Neisseria meningitidis group B
and purification of the meningococcal CMP-NeuNAc
synthetase enzyme. J Bacteriol 176: 4583–4589.
Glode MP, Robbins JB, Liu TY, Gotschlich EC, Orskov I &
Orskov F (1977) Cross-antigenicity and immunogenicity
between capsular polysaccharides of group C Neisseria
meningitidis and of Escherichia coli K92. J Infect Dis 135: 94–
104.
Goller CC & Seed PC (2010) High-throughput identification
of chemical inhibitors of E. coli Group 2 capsule biogenesis
as anti-virulence agents. PLoS ONE 5: e11642.
Granoff DM, Bartoloni A, Ricci S et al. (1998) Bactericidal
monoclonal antibodies that define unique meningococcal B
polysaccharide epitopes that do not cross-react with human
polysialic acid. J Immunol 160: 5028–5036.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
B.F. Cress et al.
Hafez M, Hayes K, Goldrick M, Warhurst G, Grencis R &
Roberts IS (2009) The K5 capsule of Escherichia coli strain
Nissle 1917 is important in mediating interactions with
intestinal epithelial cells and chemokine induction. Infect
Immun 77: 2995–3003.
Hammerschmidt S, Birkholz C, Z€ahringer U, Robertson BD,
van Putten J, Ebeling O & Frosch M (1994) Contribution of
genes from the capsule gene complex (cps) to
lipooligosaccharide biosynthesis and serum resistance in
Neisseria meningitidis. Mol Microbiol 11: 885–896.
Hancock V, Vejborg RM & Klemm P (2010) Functional
genomics of probiotic Escherichia coli Nissle 1917 and
83972, and UPEC strain CFT073: comparison of
transcriptomes, growth and biofilm formation. Mol Genet
Genomics 284: 437–454.
H€anfling P, Shashkov AS, Jann B & Jann K (1996) Analysis of
the enzymatic cleavage (beta elimination) of the capsular K5
polysaccharide of Escherichia coli by the K5-specific
coliphage: reexamination. J Bacteriol 178: 4747–4750.
Hanson BR & Neely MN (2012) Coordinate regulation of
Gram-positive cell surface components. Curr Opin Microbiol
15: 204–210.
Hanson LA, Ahlstedt S, Fasth A, Jodal U, Kaijser B, Larsson P,
Lindberg U, Olling S, Sohl-
Akerlund A & Svanborg-Eden C
(1977) Antigens of Escherichia coli, human immune
response, and the pathogenesis of urinary tract infections.
J Infect Dis 136(Suppl): S144–S149.
Harber MJ, Topley N & Asscher AW (1986) Virulence factors
of urinary pathogens. Clin Sci 70: 531–538.
Harrison OB, Claus H, Jiang Y et al. (2013) Description and
nomenclature of Neisseria meningitidis capsule locus. Emerg
Infect Dis 19: 566–573.
Hashikawa S, Iinuma Y, Furushita M, Ohkura T, Nada T,
Torii K, Hasegawa T & Ohta M (2004) Characterization of
group C and G streptococcal strains that cause
streptococcal toxic shock syndrome. J Clin Microbiol 42:
186–192.
Heidelberger M & Avery OT (1923) The soluble specific
substance of pneumococcus. J Exp Med 38: 73–79.
Herias MV, Midtvedt T, Hanson LA & Wold AE (1997)
Escherichia coli K5 capsule expression enhances colonization
of the large intestine in the gnotobiotic rat. Infect Immun
65: 531–536.
Hickey AM, Bhaskar U, Linhardt RJ & Dordick JS (2013)
Effect of eliminase gene (elmA) deletion on heparosan
production and shedding in Escherichia coli K5. J Biotechnol
165: 175–177.
Hobb RI, Tzeng YL, Choudhury BP, Carlson RW & Stephens
DS (2010) Requirement of NMB0065 for connecting
assembly and export of sialic acid capsular polysaccharides
in Neisseria meningitidis. Microbes Infect 12: 476–487.
Hodson N, Griffiths G, Cook N, Pourhossein M, Gottfridson
E, Lind T, Lidholt K & Roberts IS (2000) Identification that
KfiA, a protein essential for the biosynthesis of the
Escherichia coli K5 capsular polysaccharide, is an
alpha-UDP-GlcNAc glycosyltransferase. The formation of a
FEMS Microbiol Rev 38 (2014) 660–697
Masquerading microbial pathogens
membrane-associated K5 biosynthetic complex requires
KfiA, KfiB, and KfiC. J Biol Chem 275: 27311–27315.
€k M, Kjellen L & Johansson S (1984) Cell-surface
H€
oo
glycosaminoglycans. Annu Rev Biochem 53: 847–869.
Horwitz MA & Silverstein SC (1980) Influence of the
Escherichia coli capsule on complement fixation and on
phagocytosis and killing by human phagocytes. J Clin Invest
65: 82–94.
Huskisson E (2008) Glucosamine and Chondroitin for
Osteoarthritis. J Int Med Res 36: 1161–1179.
Jacques M, Kobisch M, Belanger M & Dugal F (1993)
Virulence of capsulated and noncapsulated isolates of
Pasteurella multocida and their adherence to porcine
respiratory tract cells and mucus. Infect Immun 61: 4785–
4792.
Jann K & Jann B (1987) Polysaccharide antigens of Escherichia
coli. Rev Infect Dis 9(Suppl 5): S517–S526.
Jann B & Jann K (1990) Structure and biosynthesis of the
capsular antigens of Escherichia coli. Curr Top Microbiol 150:
19–42.
Jann K & Jann B (1997) Capsules of Escherichia coli. Escherichia
Coli: Mechanisms of Virulence (Sussman M, ed.), pp. 113–
143. Cambridge University Press, Cambridge, UK.
Jann K & Jann B (1998) Bacterial polysaccharides related to
mammalian structures. Polysaccharides: Structural Diversity
and Functional Versatility (Dumitriu S, ed.), pp. 211–236.
Marcel Dekker, New York, NY.
Jarvis GA & Vedros NA (1987) Sialic acid of group B Neisseria
meningitidis regulates alternative complement pathway
activation. Infect Immun 55: 174–180.
Jelakovic S, Jann K & Schulz GE (1996) The three-dimensional
structure of capsule-specific CMP:
2-keto-3-deoxy-manno-octonic acid synthetase from
Escherichia coli. FEBS Lett 391: 157–161.
Jennings HJ, Roy R & Michon F (1985) Determinant
specificities of the groups B and C polysaccharides of
Neisseria meningitidis. J Immunol 134: 2651–2657.
Jensen A & Kilian M (2012) Delineation of Streptococcus
dysgalactiae, its subspecies, and its clinical and phylogenetic
relationship to Streptococcus pyogenes. J Clin Microbiol 50:
113–126.
Jiang P, Li J, Han F, Duan G, Lu X, Gu Y & Yu W (2011)
Antibiofilm activity of an exopolysaccharide from marine
bacterium Vibrio sp. QY101. PLoS ONE 6: e18514.
Jing W & DeAngelis PL (2000) Dissection of the two
transferase activities of the Pasteurella multocida hyaluronan
synthase: two active sites exist in one polypeptide.
Glycobiology 10: 883–889.
Johnson JR (1991) Virulence factors in Escherichia coli urinary
tract infection. Clin Microbiol Rev 4: 80–128.
Kabat EA, Nickerson KG, Liao J, Grossbard L, Osserman EF,
Glickman E, Chess L, Robbins JB, Schneerson R & Yang YH
(1986) A human monoclonal macroglobulin with specificity
for alpha(2—8)-linked poly-N-acetyl neuraminic acid,
the capsular polysaccharide of group B meningococci
and Escherichia coli K1, which crossreacts with
FEMS Microbiol Rev 38 (2014) 660–697
691
polynucleotides and with denatured DNA. J Exp Med 164:
642–654.
Kaijser B (1973) Immunology of Escherichia coli: K antigen
and its relation to urinary-tract infection. J Infect Dis 127:
670–677.
Kaijser B & Jodal U (1984) Escherichia coli K5 antigen in
relation to various infections and in healthy individuals.
J Clin Microbiol 19: 264–266.
Kaijser B, Hanson LA, Jodal U, Lidin-Janson G & Robbins JB
(1977) Frequency of E. coli K antigens in urinary-tract
infections in children. Lancet 1: 663–666.
Kamhi E, Joo EJ, Dordick JS & Linhardt RJ (2013)
Glycosaminoglycans in infectious disease. Biol Rev Camb
Philos 88: 928–943.
Kane TA, White CL & DeAngelis PL (2006) Functional
characterization of PmHS1, a Pasteurella multocida
heparosan synthase. J Biol Chem 281: 33192–33197.
Kaper JB, Nataro JP & Mobley HL (2004) Pathogenic
Escherichia coli. Nat Rev Microbiol 2: 123–140.
Khalil AS & Collins JJ (2010) Synthetic biology: applications
come of age. Nat Rev Genet 11: 367–379.
Kim KJ, Elliott SJ, Di Cello F, Stins MF & Kim KS (2003) The
K1 capsule modulates trafficking of E. coli-containing
vacuoles and enhances intracellular bacterial survival in
human brain microvascular endothelial cells. Cell Microbiol
5: 245–252.
Kim PJ, Lee DY, Kim TY, Lee KH, Jeong H, Lee SY & Park S
(2007) Metabolite essentiality elucidates robustness of
Escherichia coli metabolism. P Natl Acad Sci USA 104:
13638–13642.
Kim Y, Oh S & Kim SH (2009) Released exopolysaccharide
(r-EPS) produced from probiotic bacteria reduce biofilm
formation of enterohemorrhagic Escherichia coli O157:H7.
Biochem Biophys Res Commun 379: 324–329.
Kim HU, Kim SY, Jeong H, Kim TY, Kim JJ, Choy HE, Yi KY,
Rhee JH & Lee SY (2011) Integrative genome-scale
metabolic analysis of Vibrio vulnificus for drug targeting and
discovery. Mol Syst Biol 7: 1–15.
King MR, Steenbergen SM & Vimr ER (2007) Going for
baroque at the Escherichia coli K1 cell surface. Trends
Microbiol 15: 196–202.
Klainer AS & Geis I (1975) Agents of Bacterial Disease. Medical
Department, Harper & Row, New York, NY.
K€
ondgen S, Leider M, Lankester F, Bethe A, L€
ubke-Becker A,
Leendertz FH & Ewers C (2011) Pasteurella multocida
involved in respiratory disease of wild chimpanzees. PLoS
ONE 6: e24236.
Kong F, Wang W, Tao J, Wang L, Wang Q, Sabananthan A &
Gilbert GL (2005) A molecular-capsular-type prediction
system for 90 Streptococcus pneumoniae serotypes using
partial cpsA-cpsB sequencing and wzy- or wzx-specific PCR.
J Med Microbiol 54: 351–356.
Kong L, Harrington L, Li Q, Cheley S, Davis BG & Bayley H
(2013) Single-molecule interrogation of a bacterial sugar
transporter allows the discovery of an extracellular inhibitor.
Nat Chem 5: 651–659.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
692
Korhonen TK, Valtonen MV, Parkkinen J, V€ais€anen-Rhen V,
Finne J, Orskov F, Orskov I, Svenson SB & M€akel€a PH
(1985) Serotypes, hemolysin production, and receptor
recognition of Escherichia coli strains associated with
neonatal sepsis and meningitis. Infect Immun 48:
486–491.
Krahulec J, Krahulcova J, Medova M & Velebny V (2005)
Influence of KfoG on capsular polysaccharide structure in
Escherichia coli K4 strain. Mol Biotechnol 30: 129–134.
Kr€
oncke KD, Orskov I, Orskov F, Jann B & Jann K (1990)
Electron microscopic study of coexpression of adhesive
protein capsules and polysaccharide capsules in Escherichia
coli. Infect Immun 58: 2710–2714.
Kuberan B & Linhardt R (2000) Carbohydrate based vaccines.
Curr Org Chem 4: 653–677.
Kumari K & Weigel PH (1997) Molecular cloning, expression,
and characterization of the authentic hyaluronan synthase
from group C Streptococcus equisimilis. J Biol Chem 272:
32539–32546.
Lambert-Zechovsky N, Bingen E, Denamur E, Brahimi N,
Brun P, Mathieu H & Elion J (1992) Molecular analysis
provides evidence for the endogenous origin of bacteremia
and meningitis due to Enterobacter cloacae in an infant. Clin
Infect Dis 15: 30–32.
Lamppa JW, Ackerman ME, Lai JI, Scanlon TC & Griswold
KE (2011) Genetically engineered alginate lyase-PEG
conjugates exhibit enhanced catalytic function and reduced
immunoreactivity. PLoS ONE 6: e17042.
Lancefield RC (1933) A serological differentiation of human
and other groups of hemolytic streptococci. J Exp Med 57:
571–595.
Larue K, Ford RC, Willis LM & Whitfield C (2011) Functional
and structural characterization of polysaccharide
co-polymerase proteins required for polymer export in
ATP-binding cassette transporter-dependent capsule
biosynthesis pathways. J Biol Chem 286: 16658–16668.
Laurent TC & Fraser JR (1992) Hyaluronan. FASEB J 6: 2397–
2404.
Lebeer S, Vanderleyden J & De Keersmaecker SCJ (2010) Host
interactions of probiotic bacterial surface molecules:
comparison with commensals and pathogens. Nat Rev
Microbiol 8: 171–184.
Lee CJ, Banks SD & Li JP (1991) Virulence, immunity, and
vaccine related to Streptococcus pneumoniae. Crit Rev
Microbiol 18: 89–114.
Legoux R, Lelong P, Jourde C et al. (1996) N-acetyl-heparosan
lyase of Escherichia coli K5: gene cloning and expression.
J Bacteriol 178: 7260–7264.
Leying H, Suerbaum S, Kroll HP, Stahl D & Opferkuch W
(1990) The capsular polysaccharide is a major determinant
of serum resistance in K-1-positive blood culture isolates of
Escherichia coli. Infect Immun 58: 222–227.
Li L, Ly M & Linhardt RJ (2012) Proteoglycan sequence. Mol
BioSyst 8: 1613–1625.
Li Y, Hai Y, Thon V, Chen Y, Muthana MM, Qu J, Hie L &
Chen X (2013) Donor substrate promiscuity of the
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
B.F. Cress et al.
N-acetylglucosaminyltransferase activities of Pasteurella
multocida heparosan synthase 2 (PmHS2) and Escherichia
coli K5 KfiA. Appl Microbiol Biotechnol 2: 1–8.
Lidholt K & Fjelstad M (1997) Biosynthesis of the Escherichia
coli K4 capsule polysaccharide. A parallel system for studies
of glycosyltransferases in chondroitin formation. J Biol
Chem 272: 2682–2687.
Lifely MR, Nowicka UT & Moreno C (1986) Analysis of the
chain length of oligomers and polymers of sialic
acid isolated from Neisseria meningitidis group B and C and
Escherichia coli K1 and K92. Carbohydr Res 156: 123–135.
Linhardt RJ & Toida T (2004) Role of glycosaminoglycans in
cellular communication. Acc Chem Res 37: 431–438.
Linhardt RJ, Galliher PM & Cooney CL (1986) Polysaccharide
lyases. Appl Biochem Biotechnol 12: 135–176.
Liu R, Xu Y, Chen M, We€ıwer M, Zhou X, Bridges AS,
DeAngelis PL, Zhang Q, Linhardt RJ & Liu J (2010)
Chemoenzymatic design of heparan sulfate oligosaccharides.
J Biol Chem 285: 34240–34249.
Liu L, Liu Y, Li J, Du G & Chen J (2011) Microbial
production of hyaluronic acid: current state, challenges, and
perspectives. Microb Cell Fact 10: 1–9.
Lodinova-Zaadnikov
a R, Tlaskalova-Hogenova H &
Sonnenborn U (1992) Local and serum antibody response
in full–term and premature infants after artificial
colonization of the intestine with E. coli strain Nissle 1917
(Mutaflor). Pediatr Allergy Immunol 3: 43–48.
Loos M (1985) The complement system: activation and
control. Curr Top Microbiol 121: 7–18.
Lu TK & Collins JJ (2007) Dispersing biofilms with engineered
enzymatic bacteriophage. P Natl Acad Sci USA 104:
11197–11202.
Lu S, Zhang X, Zhu Y, Kim KS, Yang J & Jin Q (2011)
Complete genome sequence of the
neonatal-meningitis-associated Escherichia coli strain CE10.
J Bacteriol 193: 7005.
Luong TT & Lee CY (2002) Overproduction of type 8 capsular
polysaccharide augments Staphylococcus aureus virulence.
Infect Immun 70: 3389–3395.
Ly M, Wang Z, Laremore TN, Zhang F, Zhong W, Pu D,
Zagorevski DV, Dordick JS & Linhardt RJ (2011) Analysis
of E. coli K5 capsular polysaccharide heparosan. Anal
Bioanal Chem 399: 737–745.
Marco ML, Pavan S & Kleerebezem M (2006) Towards
understanding molecular modes of probiotic action. Curr
Opin Biotechnol 17: 204–210.
Martens EC, Roth R, Heuser JE & Gordon JI (2009) Cover
image. J Biol Chem 284(27): cover.
Masson L, Holbein BE & Ashton FE (1982) Virulence linked
to polysaccharide production in serogroup B Neisseria
meningitidis. FEMS Microbiol Lett 13: 187–190.
Mazmanian SK & Kasper DL (2006) The love-hate relationship
between bacterial polysaccharides and the host immune
system. Nat Rev Immunol 6: 849–858.
Meredith TC & Woodard RW (2006) Characterization of
Escherichia coli D-arabinose 5-phosphate isomerase encoded
FEMS Microbiol Rev 38 (2014) 660–697
Masquerading microbial pathogens
by kpsF: implications for Group 2 capsule biosynthesis.
Biochem J 395: 427–432.
Meredith TC, Mamat U, Kaczynski Z, Lindner B, Holst O &
Woodard RW (2007) Modification of lipopolysaccharide
with colanic acid (M-antigen) repeats in Escherichia coli.
J Biol Chem 282: 7790–7798.
Mergulhao FJM, Summers DK & Monteiro GA (2005)
Recombinant protein secretion in Escherichia coli. Biotechnol
Adv 23: 177–202.
Minshew BH, Jorgensen J, Swanstrum M, Grootes-Reuvecamp
GA & Falkow S (1978) Some characteristics of Escherichia
coli strains isolated from extraintestinal infections of
humans. J Infect Dis 137: 648–654.
Moe GR, Bhandari TS & Flitter BA (2009) Vaccines containing
de-N-acetyl sialic acid elicit antibodies protective against
Neisseria meningitidis groups B and C. J Immunol 182:
6610–6617.
Mohamad SAS, Azmi NA & Mohamed R (2012) Screening
and identification of Pasteurella multocida capsular types
among animal cases in Malaysia. 2012 IEEE Symposium on
Humanities, Science and Engineering Research, pp. 737–742.
IEEE, DOI: 10.1109/SHUSER.2012.6268989.
Mordhorst IL, Claus H, Ewers C et al. (2009)
O-acetyltransferase gene neuO is segregated according to
phylogenetic background and contributes to environmental
desiccation resistance in Escherichia coli K1. Environ
Microbiol 11: 3154–3165.
Moxley RA & Francis DH (1986) Natural and experimental
infection with an attaching and effacing strain of Escherichia
coli in calves. Infect Immun 53: 339–346.
Moxon ER & Kroll JS (1990) The role of bacterial
polysaccharide capsules as virulence factors. Curr Top
Microbiol 150: 65–85.
Mu~
noz R, Garcıa E & L
opez R (1998) Evidence for horizontal
transfer from Streptococcus to Escherichia coli of the kfiD
gene encoding the K5-specific UDP-glucose dehydrogenase.
J Mol Evol 46: 432–436.
Murkin AS, Chou WK, Wakarchuk WW & Tanner ME (2004)
Identification and mechanism of a bacterial hydrolyzing
UDP-N-acetylglucosamine 2-epimerase. Biochemistry 43:
14290–14298.
Mushtaq N, Redpath MB, Luzio JP & Taylor PW (2004)
Prevention and cure of systemic Escherichia coli K1 infection
by modification of the bacterial phenotype. Antimicrob
Agents Chemother 48: 1503–1508.
Navia JL, Riesenfeld J, Vann WF, Lindahl U & Roden L (1983)
Assay of N-acetylheparosan deacetylase with a capsular
polysaccharide from Escherichia coli K5 as substrate. Anal
Biochem 135: 134–140.
Ninomiya T, Sugiura N, Tawada A, Sugimoto K, Watanabe H
& Kimata K (2002) Molecular cloning and characterization
of chondroitin polymerase from Escherichia coli strain K4.
J Biol Chem 277: 21567–21575.
Nithya C, Begum MF & Pandian SK (2010) Marine bacterial
isolates inhibit biofilm formation and disrupt mature
FEMS Microbiol Rev 38 (2014) 660–697
693
biofilms of Pseudomonas aeruginosa PAO1. Appl Microbiol
Biotechnol 88: 341–358.
O’Flaherty S, Ross RP & Coffey A (2009) Bacteriophage and
their lysins for elimination of infectious bacteria. FEMS
Microbiol Rev 33: 801–819.
O’Hanlon KA, Catarame TMG, Duffy G, Blair IS & McDowell
DA (2004) RAPID detection and quantification of E. coli
O157/O26/O111 in minced beef by real-time PCR. J Appl
Microbiol 96: 1013–1023.
Orskov F & Orskov I (1992) Escherichia coli serotyping and
disease in man and animals. Can J Microbiol 38: 699–704.
Orskov I, Orskov F, Jann B & Jann K (1977) Serology,
chemistry, and genetics of O and K antigens of Escherichia
coli. Bacteriol Rev 41: 667–710.
Orskov F, Orskov I, Sutton A, Schneerson R, Lin W, Egan W,
Hoff GE & Robbins JB (1979) Form variation in Escherichia
coli K1: determined by O-acetylation of the capsular
polysaccharide. J Exp Med 149: 669–685.
Orskov I, Birch-Andersen A, Duguid JP, Stenderup J & Orskov
F (1985) An adhesive protein capsule of Escherichia coli.
Infect Immun 47: 191–200.
Orth JD, Thiele I & Palsson BØ (2010) What is flux balance
analysis? Nat Biotechnol 28: 245–248.
Osawa T, Sugiura N, Shimada H, Hirooka R, Tsuji A,
Shirakawa T, Fukuyama K, Kimura M, Kimata K & Kakuta
Y (2009) Crystal structure of chondroitin polymerase from
Escherichia coli K4. Biochem Biophys Res Commun 378: 10–
14.
van Oss CJ, Gillman CF & Neumann AW (1975) Phagocytic
Engulfment and Cell Adhesiveness as Cellular Surface
Phenomena. Vol. 2. Marcel Dekker, New York and Basel.
Ostblom A, Adlerberth I, Wold AE & Nowrouzian FL (2011)
Pathogenicity island markers, virulence determinants malX
and usp, and the capacity of Escherichia coli to persist in
infants’ commensal microbiotas. Appl Environ Microbiol 77:
2303–2308.
Otto NJ, Green DE, Masuko S, Mayer A, Tanner ME, Linhardt
RJ & DeAngelis PL (2012) Structure/function analysis of
Pasteurella multocida heparosan synthases: toward defining
enzyme specificity and engineering novel catalysts. J Biol
Chem 287: 7203–7212.
Pavelka MS, Wright LF & Silver RP (1991) Identification of two
genes, kpsM and kpsT, in region 3 of the polysialic acid gene
cluster of Escherichia coli K1. J Bacteriol 173: 4603–4610.
Pavelka MS, Hayes SF & Silver RP (1994) Characterization of
KpsT, the ATP-binding component of the ABC-transporter
involved with the export of capsular polysialic acid in
Escherichia coli K1. J Biol Chem 269: 20149–20158.
Pazzani C, Rosenow C, Boulnois GJ, Bronner D, Jann K &
Roberts IS (1993) Molecular analysis of region 1 of the
Escherichia coli K5 antigen gene cluster: a region encoding
proteins involved in cell surface expression of capsular
polysaccharide. J Bacteriol 175: 5978–5983.
Peterson PK, Wilkinson BJ, Kim Y, Schmeling D & Quie PG
(1978) Influence of encapsulation on staphylococcal
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
694
opsonization and phagocytosis by human
polymorphonuclear leukocytes. Infect Immun 19: 943–949.
Petit C, Rigg GP, Pazzani C, Smith A, Sieberth V, Stevens M,
Boulnois G, Jann K & Roberts IS (1995) Region 2 of the
Escherichia coli K5 capsule gene cluster encoding proteins
for the biosynthesis of the K5 polysaccharide. Mol Microbiol
17: 611–620.
Pigeon RP & Silver RP (1994) Topological and mutational
analysis of KpsM, the hydrophobic component of the
ABC-transporter involved in the export of polysialic acid in
Escherichia coli K1. Mol Microbiol 14: 871–881.
Pluschke G, Mayden J, Achtman M & Levine RP (1983) Role
of the capsule and the O antigen in resistance of O18:K1
Escherichia coli to complement-mediated killing. Infect
Immun 42: 907–913.
Poermadjaja B & Frost A (2000) Phagocytic uptake and killing
of virulent and avirulent strains of Pasteurella multocida of
capsular serotype A by chicken macrophages. Vet Microbiol
72: 163–171.
Pon RA, Lussier M, Yang QL & Jennings HJ (1997)
N-Propionylated group B meningococcal polysaccharide
mimics a unique bactericidal capsular epitope in group B
Neisseria meningitidis. J Exp Med 185: 1929–1938.
Prasad SB, Jayaraman G & Ramachandran KB (2010)
Hyaluronic acid production is enhanced by the additional
co-expression of UDP-glucose pyrophosphorylase in
Lactococcus lactis. Appl Microbiol Biotechnol 86: 273–283.
Rafiq I, Parthasarathy H, Tremlett C, Freeman LJ & Mullin M
(2011) Infective endocarditis caused by Moraxella
nonliquefaciens in a percutaneous aortic valve replacement.
Cardiovasc Revasc Med 12: 184–186.
Rahn A, Beis K, Naismith JH & Whitfield C (2003) A novel
outer membrane protein, Wzi, is involved in surface
assembly of the Escherichia coli K30 Group 1 capsule.
J Bacteriol 185: 5882–5890.
Raj A & van Oudenaarden A (2009) Single-molecule
approaches to stochastic gene expression. Annu Rev Biophys
38: 255–270.
Ralph AP & Carapetis JR (2013) Group a streptococcal diseases
and their global burden. Curr Top Microbiol 368: 1–27.
Reid AN & Cuthbertson L (2012) Biosynthesis of capsular
polysaccharides and exopolysaccharides. Bacterial Glycomics:
Current Research, Technology and Applications (Reid CW,
Twine SM & Reid AN, eds), pp. 27–54. Caister Academic
Press, Norfolk, UK.
Reizer J, Reizer A & Saier MH (1992) A new subfamily of
bacterial ABC-type transport systems catalyzing export of
drugs and carbohydrates. Protein Sci 1: 1326–1332.
Restaino OF, Cimini D, De Rosa M, Catapano A & Schiraldi C
(2011) High cell density cultivation of Escherichia coli K4 in a
microfiltration bioreactor: a step towards improvement of
chondroitin precursor production. Microb Cell Fact 10: 1–10.
Restaino OF, Di Lauro I, Cimini D, Carlino E, De Rosa M &
Schiraldi C (2012) Monosaccharide precursors for boosting
chondroitin-like capsular polysaccharide production. Appl
Microbiol Biotechnol 97: 1699–1709.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
B.F. Cress et al.
Rice JA, Carrasco-Medina L, Hodgins DC & Shewen PE
(2007) Mannheimia haemolytica and bovine respiratory
disease. Anim Health Res Rev 8: 117–128.
Rigg GP, Barrett B & Roberts IS (1998) The localization of
KpsC, S and T, and KfiA, C and D proteins involved in the
biosynthesis of the Escherichia coli K5 capsular
polysaccharide: evidence for a membrane-bound complex.
Microbiology 144(Pt 10): 2905–2914.
Rimler RB & Rhoades KR (1987) Serogroup F, a new capsule
serogroup of Pasteurella multocida. J Clin Microbiol 25: 615–
618.
Rimler RB, Register KB, Magyar T & Ackermann MR (1995)
Influence of chondroitinase on indirect hemagglutination
titers and phagocytosis of Pasteurella multocida serogroups
A, D and F. Vet Microbiol 47: 287–294.
Robbins JB, McCracken GH, Gotschlich EC, Orskov F, Orskov
I & Hanson LA (1974) Escherichia coli K1 capsular
polysaccharide associated with neonatal meningitis. N Engl J
Med 290: 1216–1220.
Roberts IS (1996) The biochemistry and genetics of capsular
polysaccharide production in bacteria. Annu Rev Microbiol
50: 285–315.
Roberts IS, Mountford R, High N, Bitter-Suermann D, Jann K,
Timmis K & Boulnois G (1986) Molecular cloning and
analysis of genes for production of K5, K7, K12, and K92
capsular polysaccharides in Escherichia coli. J Bacteriol 168:
1228–1233.
Rodriguez ML, Jann B & Jann K (1988) Structure and
serological characteristics of the capsular K4 antigen of
Escherichia coli O5:K4:H4, a fructose-containing
polysaccharide with a chondroitin backbone. Eur J Biochem
177: 117–124.
Rosenow C, Roberts IS & Jann K (1995a) Isolation from
recombinant Escherichia coli and characterization of
CMP-Kdo synthetase, involved in the expression of the
capsular K5 polysaccharide (K-CKS). FEMS Microbiol Lett
125: 159–164.
Rosenow C, Esumeh F, Roberts IS & Jann K (1995b)
Characterization and localization of the KpsE protein of
Escherichia coli K5, which is involved in polysaccharide
export. J Bacteriol 177: 1137–1143.
Rowe S, Hodson N, Griffiths G & Roberts IS (2000)
Regulation of the Escherichia coli K5 capsule gene cluster:
evidence for the roles of H-NS, BipA, and integration host
factor in regulation of Group 2 capsule gene clusters in
pathogenic E. coli. J Bacteriol 182: 2741–2745.
Rutishauser U (2008) Polysialic acid in the plasticity of the
developing and adult vertebrate nervous system. Nat Rev
Neurosci 9: 26–35.
Rutishauser U, Watanabe M, Silver J, Troy FA & Vimr ER
(1985) Specific alteration of NCAM-mediated cell
adhesion by an endoneuraminidase. J Cell Biol 101:
1842–1849.
Sandberg T, Kaijser B, Lidin-Janson G, Lincoln K, Orskov F,
Orskov I, Stokland E & Svanborg-Eden C (1988) Virulence
of Escherichia coli in relation to host factors in women with
FEMS Microbiol Rev 38 (2014) 660–697
Masquerading microbial pathogens
symptomatic urinary tract infection. J Clin Microbiol 26:
1471–1476.
Sarff LD, McCracken GH, Schiffer MS, Glode MP, Robbins JB,
Orskov I & Orskov F (1975) Epidemiology of Escherichia
coli K1 in healthy and diseased newborns. Lancet 1: 1099–
1104.
Sawata A & Kume K (1983) Relationships between virulence
and morphological or serological properties of variants
dissociated from serotype 1 Haemophilus paragallinarum
strains. J Clin Microbiol 18: 49–55.
Schiraldi C, Cimini D & De Rosa M (2010) Production of
chondroitin sulfate and chondroitin. Appl Microbiol
Biotechnol 87: 1209–1220.
Schiraldi C, Carcarino IL, Alfano A, Restaino OF, Panariello A
& De Rosa M (2011) Purification of chondroitin precursor
from Escherichia coli K4 fermentation broth using
membrane processing. Biotechnol J 6: 410–419.
Schiraldi C, Alfano A, Cimini D, De Rosa M, Panariello A &
Restaino Odile F (2012) Application of a 22L scale
membrane bioreactor and cross-flow ultrafiltration to obtain
purified chondroitin. Biotechnol Prog 28: 1012–1018.
Scholl D, Rogers S, Adhya S & Merril CR (2001)
Bacteriophage K1-5 encodes two different tail fiber proteins,
allowing it to infect and replicate on both K1 and K5 strains
of Escherichia coli. J Virol 75: 2509–2515.
Scholl D, Adhya S & Merril C (2005) Escherichia coli K1’s
capsule is a barrier to bacteriophage T7. Appl Environ
Microbiol 71: 4872–4874.
Schrager HM, Rheinwald JG & Wessels MR (1996) Hyaluronic
acid capsule and the role of streptococcal entry into
keratinocytes in invasive skin infection. J Clin Invest 98:
1954–1958.
Schroeder B, Duncker S, Barth S, Bauerfeind R, Gruber AD,
Deppenmeier S & Breves G (2006) Preventive effects of the
probiotic Escherichia coli strain Nissle 1917 on acute
secretory diarrhea in a pig model of intestinal infection. Dig
Dis Sci 51: 724–731.
Schulz EC, Bergfeld AK, Ficner R & M€
uhlenhoff M (2011)
Crystal structure analysis of the polysialic acid specific
O-acetyltransferase NeuO. PLoS ONE 6: e17403.
Shen Y, Liu J, Estiu G, Isin B, Ahn YY, Lee DS, Barabasi AL,
Kapatral V, Wiest O & Oltvai ZN (2010) Blueprint for
antimicrobial hit discovery targeting metabolic networks.
P Natl Acad Sci USA 107: 1082–1087.
Shuren J (2006) Food additives permitted for direct addition
to food for human consumption; bacteriophage preparation.
Fed Reg 71: 47729–47732.
Sieberth V, Rigg GP, Roberts IS & Jann K (1995) Expression
and characterization of UDPGlc dehydrogenase (KfiD),
which is encoded in the type-specific region 2 of the
Escherichia coli K5 capsule genes. J Bacteriol 177: 4562–4565.
Silver RP, Finn CW, Vann WF, Aaronson W, Schneerson R,
Kretschmer PJ & Garon CF (1981) Molecular cloning of the
K1 capsular polysaccharide genes of E. coli. Nature 289:
696–698.
FEMS Microbiol Rev 38 (2014) 660–697
695
Silver RP, Aaronson W & Vann WF (1988) The K1 capsular
polysaccharide of Escherichia coli. Rev Infect Dis 10(Suppl 2):
S282–S286.
Simonen M & Palva I (1993) Protein secretion in Bacillus
species. Microbiol Rev 57: 109–137.
Simpson DA, Hammarton TC & Roberts IS (1996)
Transcriptional organization and regulation of expression of
region 1 of the Escherichia coli K5 capsule gene cluster.
J Bacteriol 178: 6466–6474.
Smith AN, Boulnois GJ & Roberts IS (1990) Molecular
analysis of the Escherichia coli K5 kps locus: identification
and characterization of an inner-membrane capsular
polysaccharide transport system. Mol Microbiol 4: 1863–
1869.
Spinosa MR, Progida C, Tala A, Cogli L, Alifano P & Bucci C
(2007) The Neisseria meningitidis capsule is important for
intracellular survival in human cells. Infect Immun 75: 3594–
3603.
Steen JA, Steen JA, Harrison P, Seemann T, Wilkie I, Harper
M, Adler B & Boyce JD (2010) Fis is essential for capsule
production in Pasteurella multocida and regulates expression
of other important virulence factors. PLoS Pathog 6:
e1000750.
Steenbergen SM & Vimr ER (2008) Biosynthesis of the
Escherichia coli K1 Group 2 polysialic acid capsule occurs
within a protected cytoplasmic compartment. Mol Microbiol
68: 1252–1267.
Stevens P, Huang SN, Welch WD & Young LS (1978)
Restricted complement activation by Escherichia coli with
the K-1 capsular serotype: a possible role in pathogenicity.
J Immunol 121: 2174–2180.
Stevens MP, Clarke BR & Roberts IS (1997) Regulation of the
Escherichia coli K5 capsule gene cluster by transcription
antitermination. Mol Microbiol 24: 1001–1012.
Stordeur P, China B, Charlier G, Roels S & Mainil J (2000)
Clinical signs, reproduction of attaching/effacing lesions,
and enterocyte invasion after oral inoculation of an O118
enterohaemorrhagic Escherichia coli in neonatal calves.
Microbes Infect 2: 17–24.
Storm DW, Koff SA, Horvath DJ, Li B & Justice SS (2011) In
vitro analysis of the bactericidal activity of Escherichia coli
Nissle 1917 against pediatric uropathogens. J Urol 186:
1678–1683.
Stummeyer K, Dickmanns A, M€
uhlenhoff M, Gerardy-Schahn
R & Ficner R (2004) Crystal structure of the polysialic acid–
degrading endosialidase of bacteriophage K1F. Nat Struct
Mol Biol 12: 90–96.
Sugahara K & Mikami T (2007) Chondroitin/dermatan sulfate
in the central nervous system. Curr Opin Struct Biol 17:
536–545.
Sulakvelidze A, Alavidze Z & Morris JG (2001) Bacteriophage
therapy. Antimicrob Agents Chemother 45: 649–659.
Sun J, Gunzer F, Westendorf AM, Buer J, Scharfe M, Jarek M,
G€
ossling F, Bl€
ocker H & Zeng AP (2005) Genomic
peculiarity of coding sequences and metabolic potential of
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
696
probiotic Escherichia coli strain Nissle 1917 inferred from
raw genome data. J Biotechnol 117: 147–161.
Sun W, Du L & Li M (2010) Aptamer-based carbohydrate
recognition. Curr Pharm Des 16: 2269–2278.
Surana NK & Kasper DL (2012) The yin yang of bacterial
polysaccharides: lessons learned from B. fragilis PSA.
Immunol Rev 245: 13–26.
Tang L, Liu Y, Ali MM, Kang DK, Zhao W & Li J (2012)
Colorimetric and ultrasensitive bioassay based on a
dual-amplification system using aptamer and DNAzyme.
Anal Chem 84: 4711–4717.
Thelin MA, Bartolini B, Axelsson J, Gustafsson R, Tykesson E,
Pera E, Oldberg A, Maccarana M & Malmstrom A (2013)
Biological functions of iduronic acid in chondroitin/
dermatan sulfate. FEBS J 280: 2431–2446.
Thompson JE, Pourhossein M, Waterhouse A, Hudson T,
Goldrick M, Derrick JP & Roberts IS (2010) The K5 lyase
KflA combines a viral tail spike structure with a bacterial
polysaccharide lyase mechanism. J Biol Chem 285: 23963–
23969.
Tian X, Azpurua J, Hine C, Vaidya A, Myakishev-Rempel M,
Ablaeva J, Mao Z, Nevo E, Gorbunova V & Seluanov A
(2013) High-molecular-mass hyaluronan mediates the
cancer resistance of the naked mole rat. Nature 499: 346–
349.
Tjalsma H, Antelmann H, Jongbloed JD et al. (2004)
Proteomics of protein secretion by Bacillus subtilis:
separating the “secrets” of the secretome. Microbiol Mol Biol
Rev 68: 207–233.
Tombelli S, Minunni M & Mascini M (2005) Analytical
applications of aptamers. Biosens Bioelectron 20: 2424–2434.
Townsend KM, Boyce JD, Chung JY, Frost AJ & Adler B
(2001) Genetic organization of Pasteurella multocida cap
Loci and development of a multiplex capsular PCR typing
system. J Clin Microbiol 39: 924–929.
Trilli A, Busiello I, Daly S & Bagatin F (2012) Biotechnological
production of chondroitin. WIPO Patent Number
2012004063.
Troy FA (1992) Polysialylation: from bacteria to brains.
Glycobiology 2: 5–23.
Tzeng Y, Datta AK, Strole CA, Lobritz MA, Carlson RW &
Stephens DS (2005) Translocation and surface expression of
lipidated serogroup B capsular polysaccharide in Neisseria
meningitidis. Infect Immun 73: 1491–1505.
Valle J, Da Re S, Henry N, Fontaine T, Balestrino D,
Latour-Lambert P & Ghigo JM (2006) Broad-spectrum
biofilm inhibition by a secreted bacterial polysaccharide. P
Natl Acad Sci USA 103: 12558–12563.
Vann WF, Schmidt MA, Jann B & Jann K (1981) The
structure of the capsular polysaccharide (K5 antigen) of
urinary-tract-infective Escherichia coli 010:K5:H4. A polymer
similar to desulfo-heparin. Eur J Biochem 116: 359–364.
Vann WF, Daines DA, Murkin AS, Tanner ME, Chaffin DO,
Rubens CE, Vionnet J & Silver RP (2004) The NeuC protein
of Escherichia coli K1 is a UDP N-acetylglucosamine
2-epimerase. J Bacteriol 186: 706–712.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
B.F. Cress et al.
Varki A (2006) Nothing in glycobiology makes sense, except in
the light of evolution. Cell 126: 841–845.
Veasy LG, Tani LY, Daly JA, Korgenski K, Miner L, Bale J,
Kaplan EL, Musser JM & Hill HR (2004) Temporal
association of the appearance of mucoid strains of
Streptococcus pyogenes with a continuing high incidence of
rheumatic fever in Utah. Pediatrics 113: e168–e172.
Vimr ER & Steenbergen SM (2006) Mobile contingency locus
controlling Escherichia coli K1 polysialic acid capsule
acetylation. Mol Microbiol 60: 828–837.
Vimr ER, Aaronson W & Silver RP (1989) Genetic analysis of
chromosomal mutations in the polysialic acid gene cluster
of Escherichia coli K1. J Bacteriol 171: 1106–1117.
Vinkenborg JL, Karnowski N & Famulok M (2011) Aptamers
for allosteric regulation. Nat Chem Biol 7: 519–527.
Vinti~
ni E, Villena J, Alvarez S & Medina M (2010)
Administration of a probiotic associated with nasal
vaccination with inactivated Lactococcus lactis-PppA
induces effective protection against pneumoccocal infection
in young mice. Clin Exp Immunol 159: 351–362.
Voigt CA (2006) Genetic parts to program bacteria. Curr Opin
Biotechnol 17: 548–557.
Vollmer W (2008) Structural variation in the glycan strands of
bacterial peptidoglycan. FEMS Microbiol Rev 32: 287–306.
Volpi N (2007) Mass spectrometry characterization of
Escherichia coli K4 oligosaccharides from 2-mers to more
than 20-mers. Rapid Commun Mass Spectrom 21: 3459–
3466.
Volpi N, Zhang Z & Linhardt RJ (2008) Mass spectrometry for
the characterization of unsulfated chondroitin
oligosaccharides from 2-mers to 16-mers. Comparison with
hyaluronic acid oligomers. Rapid Commun Mass Spectrom
22: 3526–3530.
Wang X, Preston JF & Romeo T (2004) The pgaABCD locus
of Escherichia coli promotes the synthesis of a polysaccharide
adhesin required for biofilm formation. J Bacteriol 186:
2724–2734.
Wang X, Dubey AK, Suzuki K, Baker CS, Babitzke P & Romeo
T (2005) CsrA post-transcriptionally represses pgaABCD,
responsible for synthesis of a biofilm polysaccharide adhesin
of Escherichia coli. Mol Microbiol 56: 1648–1663.
Wang Z, Ly M, Zhang F, Zhong W, Suen A, Hickey AM,
Dordick JS & Linhardt RJ (2010) E. coli K5 fermentation
and the preparation of heparosan, a bioengineered heparin
precursor. Biotechnol Bioeng 107: 964–973.
Wang Z, Yang B, Zhang Z, Ly M, Takieddin M, Mousa S, Liu
J, Dordick JS & Linhardt RJ (2011) Control of the
heparosan N-deacetylation leads to an improved
bioengineered heparin. Appl Microbiol Biotechnol 91: 91–99.
Ward PN, Field TR, Ditcham WG, Maguin E & Leigh JA
(2001) Identification and disruption of two discrete loci
encoding hyaluronic acid capsule biosynthesis genes hasA,
hasB, and hasC in Streptococcus uberis. Infect Immun 69:
392–399.
Watt JM, Wade MM, Holman SC, Wilson WW, Keil DE,
Pruett SB, Jacques M & Champlin FR (2003) Influence of
FEMS Microbiol Rev 38 (2014) 660–697
Masquerading microbial pathogens
serotype A capsulation on cell surface physiologic factors in
Pasteurella multocida. Colloids Surf B 28: 227–238.
Wei Z, Fu Q, Chen Y, Cong P, Xiao S, Mo D, He Z & Liu X
(2012) The capsule of Streptococcus equi ssp. zooepidemicus is
a target for attenuation in vaccine development. Vaccine 30:
4670–4675.
Weintraub A (2003) Immunology of bacterial polysaccharide
antigens. Carbohydr Res 338: 2539–2547.
Wells CL, Maddaus MA & Simmons RL (1988) Proposed
mechanisms for the translocation of intestinal bacteria. Rev
Infect Dis 10: 958–979.
Wessels MR, Rubens CE, Benedı VJ & Kasper DL (1989)
Definition of a bacterial virulence factor: sialylation of the
group B streptococcal capsule. P Natl Acad Sci USA 86:
8983–8987.
Wessels MR, Moses AE, Goldberg JB & DiCesare TJ (1991)
Hyaluronic acid capsule is a virulence factor for mucoid
group A streptococci. P Natl Acad Sci USA 88: 8317–8321.
Whitfield C (2006) Biosynthesis and assembly of capsular
polysaccharides in Escherichia coli. Annu Rev Biochem 75:
39–68.
Whitfield C & Roberts IS (1999) Structure, assembly and
regulation of expression of capsules in Escherichia coli. Mol
Microbiol 31: 1307–1319.
Wibawan IW, Pasaribu FH, Utama IH, Abdulmawjood A &
L€ammler C (1999) The role of hyaluronic acid capsular
material of Streptococcus equi subsp. zooepidemicus in
mediating adherence to HeLa cells and in resisting
phagocytosis. Res Vet Sci 67: 131–135.
Widner B, Behr R, Von Dollen S, Tang M, Heu T, Sloma A,
Sternberg D, DeAngelis PL, Weigel PH & Brown S (2005)
Hyaluronic acid production in Bacillus subtilis. Appl Environ
Microbiol 71: 3747–3752.
Wildi LM, Raynauld JP, Martel-Pelletier J, Beaulieu A, Bessette
L, Morin F, Abram F, Dorais M & Pelletier JP (2011)
Chondroitin sulphate reduces both cartilage volume loss
and bone marrow lesions in knee osteoarthritis patients
starting as early as 6 months after initiation of therapy: a
randomised, double-blind, placebo-controlled pilot study
using MRI. Ann Rheum Dis 70: 982–989.
Wiles TJ, Kulesus RR & Mulvey MA (2008) Origins and
virulence mechanisms of uropathogenic Escherichia coli. Exp
Mol Pathol 85: 11–19.
Willis LM, Stupak J, Richards MR, Lowary TL, Li J &
Whitfield C (2013) Conserved glycolipid termini in capsular
polysaccharides synthesized by ATP-binding cassette
transporter-dependent pathways in Gram-negative
pathogens. P Natl Acad Sci USA 110: 7868–7873.
Winkelstein JA (1981) The role of complement in the host’s
defense against Streptococcus pneumoniae. Rev Infect Dis 3:
289–298.
Wu JR, Chen PY, Shien JH, Shyu CL, Shieh HK, Chang F &
Chang PC (2010) Analysis of the biosynthesis genes and
chemical components of the capsule of Avibacterium
paragallinarum. Vet Microbiol 145: 90–99.
FEMS Microbiol Rev 38 (2014) 660–697
697
Wu Q, Yang A, Zou W, Duan Z, Liu J, Chen J & Liu L (2013)
Transcriptional engineering of Escherichia coli K4 for
fructosylated chondroitin production. Biotechnol Prog 29:
1140–1149.
Wunder DE, Aaronson W, Hayes SF, Bliss JM & Silver RP
(1994) Nucleotide sequence and mutational analysis of the
gene encoding KpsD, a periplasmic protein involved in
transport of polysialic acid in Escherichia coli K1. J Bacteriol
176: 4025–4033.
Xue P, Corbett D, Goldrick M, Naylor C & Roberts IS (2009)
Regulation of expression of the region 3 promoter of the
Escherichia coli K5 capsule gene cluster involves H-NS, SlyA,
and a large 5′ untranslated region. J Bacteriol 191:
1838–1846.
Yi W, Zhu L, Guo H, Mei L, Li J & Wang PG (2006)
Formation of a new O-polysaccharide in Escherichia coli
O86 via disruption of a glycosyltransferase gene involved in
O-unit assembly. Carbohydr Res 341: 2254–2260.
Yu H & Stephanopoulos G (2008) Metabolic engineering of
Escherichia coli for biosynthesis of hyaluronic acid. Metab
Eng 10: 24–32.
Yu H, Tyo K, Alper H, Klein-Marcuschamer D &
Stephanopoulos G (2008) A high-throughput screen for
hyaluronic acid accumulation in recombinant Escherichia
coli transformed by libraries of engineered sigma factors.
Biotechnol Bioeng 101: 788–796.
Zanfardino A, Restaino OF, Notomista E, Cimini D, Schiraldi C,
De Rosa M, De Felice M & Varcamonti M (2010) Isolation of
an Escherichia coli K4 kfoC mutant over-producing capsular
chondroitin. Microb Cell Fact 9: 1–8.
Zehnder JL & Galli SJ (1999) Mast-cell heparin demystified.
Nature 400: 714–715.
Zhang C, Liu L, Teng L, Chen J, Liu J, Li J, Du G & Chen J
(2012) Metabolic engineering of Escherichia coli BL21 for
biosynthesis of heparosan, a bioengineered heparin
precursor. Metab Eng 14: 521–527.
Zhao Q, Sun Y, Zhang X, Kong Y, Xie Z, Zhu Y, Zhou E &
Jiang S (2010) Evaluation of two experimental infection
models for Avibacterium paragallinarum. Vet Microbiol 141:
68–72.
Zhu H, Wang Q, Wen L, Xu J, Shao Z, Chen M, Chen M,
Reeves PR, Cao B & Wang L (2012) Development of a
multiplex PCR assay for detection and genogrouping of
Neisseria meningitidis. J Clin Microbiol 50: 46–51.
Zingler G, Schmidt G, Orskov I, Orskov F, Falkenhagen U &
Naumann G (1990) K-antigen identification, hemolysin
production, and hemagglutination types of Escherichia coli
O6 strains isolated from patients with urinary tract
infections. Zentralbl Bakteriol 274: 372–381.
Zogaj X, Nimtz M, Rohde M, Bokranz W & R€
omling U
(2001) The multicellular morphotypes of Salmonella
typhimurium and Escherichia coli produce cellulose as the
second component of the extracellular matrix. Mol Microbiol
39: 1452–1463.
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